Compositions And Methods For Improving Post-Harvest Properties Of Agricultural Crops

ABSTRACT

The present invention relates to methods for modifying an agricultural crop comprising treating the agricultural crop with a composition comprising a xyloglucan endotransglycosylase and (a) a polymeric xyloglucan and a functionalized xyloglucan oligomer comprising a chemical group; (b) a polymeric xyloglucan functionalized with a chemical group and a functionalized xyloglucan oligomer comprising a chemical group; (c) a polymeric xyloglucan functionalized with a chemical group and a xyloglucan oligomer; (d) a polymeric xyloglucan, and a xyloglucan oligomer; (e) a polymeric xyloglucan functionalized with a chemical group; (f) a polymeric xyloglucan; (g) a functionalized xyloglucan oligomer comprising a chemical group; or (h) a xyloglucan oligomer, or (a-h) without a xyloglucan endotransglycosylase, in a medium under conditions leading to a modified agricultural crop possessing an improved property compared to the unmodified agricultural crop. The present invention also relates to a modified agricultural crop obtained by such methods.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional application of U.S. application Ser.No. 15/122,610 filed on Aug. 30, 2016, which is a 35 U.S.C. § 371national application of PCT/US2015/019011 filed on Mar. 5, 2015, whichclaims priority or the benefit under 35 U.S.C. § 119 of U.S. ProvisionalApplication No. 61/948,232 filed on Mar. 5, 2014, the contents of whichare fully incorporated herein by reference.

REFERENCE TO A SEQUENCE LISTING

This application contains a Sequence Listing in computer readable form,which is incorporated herein by reference.

BACKGROUND OF THE INVENTION Field of the Invention

The present invention relates to compositions and methods for improvingproperties of agricultural crops.

Description of the Related Art

Xyloglucan endotransglycosylase (XET) is an enzyme that catalyzesendotransglycosylation of xyloglucan, a structural polysaccharide ofplant cell walls. The enzyme is present in most plants, and inparticular, land plants. XET has been extracted from dicotyledons andmonocotyledons.

Xyloglucan is present in cotton, paper, or wood fibers (Hayashi et al.,1988, Carbohydrate Research 181: 273-277) making strong hydrogen bondsto cellulose (Carpita and Gibeaut, 1993, The Plant Journal 3: 1-30).Adding xyloglucan endotransglycosylase to various cellulosic materialscontaining xyloglucan alters the xyloglucan mediated interlinkagesbetween cellulosic fibers improving their strength, and maintaining thecellulose-structure while permitting the cellulose fibers to moverelative to one another under force.

It is known in the art that much of the agricultural crops grown ingreenhouses and particularly open fields is spoiled by exposure to theenvironment or to agricultural pests. It is desirable in the art to formphysical protection or barriers around agricultural crops without theuse of chemical or biological pesticides. U.S. Pat. No. 6,027,740; U.S.Pat. No. 6,069,112; U.S. Pat. No. 6,110,867, and U.S. Pat. No. 6,156,327disclose methods of crop protection by generating a physical barrieraround produce. It is also known that much of the produce harvested fromfields, gardens and greenhouses is lost to spoilage before consumptionor sale. The quantity of loss is estimated from 0 to 25% in first worldnations, and 0 to 50% in third world nations, depending on the cropharvested, which extrapolates to substantial economic, nutritive andsociological loss. In first world nations, the majority of post-harvestloss is termed qualitative loss; produce not spoiled remains unconsumedor unsold due to negative appearance.

There is a need in the art to preserve agricultural crops, both inappearance and from spoilage, rot, or contamination. There is also aneed in the art to extend the length of time between harvest and marketover which harvested crops remain fresh in appearance. There is afurther need in the art to preserve or slow the onset of spoilage or theappearance of spoilage for cut or prepared produce.

The present invention provides methods for improving properties ofagricultural crops.

SUMMARY OF THE INVENTION

The present invention relates to methods for modifying an agriculturalcrop comprising treating the agricultural crop with a compositionselected from the group consisting of (a) a composition comprising axyloglucan endotransglycosylase, a polymeric xyloglucan, and afunctionalized xyloglucan oligomer comprising a chemical group; (b) acomposition comprising a xyloglucan endotransglycosylase, a polymericxyloglucan functionalized with a chemical group, and a functionalizedxyloglucan oligomer comprising a chemical group; (c) a compositioncomprising a xyloglucan endotransglycosylase, a polymeric xyloglucanfunctionalized with a chemical group, and a xyloglucan oligomer; (d) acomposition comprising a xyloglucan endotransglycosylase, a polymericxyloglucan, and a xyloglucan oligomer; (e) a composition comprising axyloglucan endotransglycosylase and a polymeric xyloglucanfunctionalized with a chemical group; (f) a composition comprising axyloglucan endotransglycosylase and a polymeric xyloglucan; (g) acomposition comprising a xyloglucan endotransglycosylase and afunctionalized xyloglucan oligomer comprising a chemical group; (h) acomposition comprising a xyloglucan endotransglycosylase and axyloglucan oligomer, and (i) a composition of (a), (b), (c), (d), (e),(f), (g), or (h) without a xyloglucan endotransglycosylase, wherein themodified agricultural crop possesses an improved property compared tothe unmodified agricultural crop.

The present invention also relates to modified agricultural cropsobtained by such methods.

The present invention also relates to modified agricultural cropscomprising (a) a polymeric xyloglucan and a functionalized xyloglucanoligomer comprising a chemical group; (b) a polymeric xyloglucanfunctionalized with a chemical group and a functionalized xyloglucanoligomer comprising a chemical group; (c) a polymeric xyloglucanfunctionalized with a chemical group and a xyloglucan oligomer; (d) apolymeric xyloglucan and a xyloglucan oligomer; (e) a polymericxyloglucan functionalized with a chemical group; (f) a polymericxyloglucan; (g) a functionalized xyloglucan oligomer comprising achemical group; or (h) a xyloglucan oligomer, wherein the modifiedagricultural crop possesses an improved property compared to theunmodified agricultural crop.

The present invention further relates to a composition selected from thegroup consisting of (a) a composition comprising a xyloglucanendotransglycosylase, a polymeric xyloglucan, and a functionalizedxyloglucan oligomer comprising a chemical group; (b) a compositioncomprising a xyloglucan endotransglycosylase, a polymeric xyloglucanfunctionalized with a chemical group, and a functionalized xyloglucanoligomer comprising a chemical group; (c) a composition comprising axyloglucan endotransglycosylase, a polymeric xyloglucan functionalizedwith a chemical group, and a xyloglucan oligomer; (d) a compositioncomprising a xyloglucan endotransglycosylase, a polymeric xyloglucan,and a xyloglucan oligomer; (e) a composition comprising a xyloglucanendotransglycosylase and a polymeric xyloglucan functionalized with achemical group; (f) a composition comprising a xyloglucanendotransglycosylase and a polymeric xyloglucan; (g) a compositioncomprising a xyloglucan endotransglycosylase and a functionalizedxyloglucan oligomer comprising a chemical group; (h) a compositioncomprising a xyloglucan endotransglycosylase and a xyloglucan oligomer,and (i) a composition of (a), (b), (c), (d), (e), (f), (g), or (h)without a xyloglucan endotransglycosylase.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows a restriction map of pDLHD0012.

FIG. 2 shows a restriction map of pMMar27.

FIG. 3 shows a restriction map of pEvFz1.

FIG. 4 shows a restriction map of pDLHD0006.

FIG. 5 shows a restriction map of pDLHD0039.

FIG. 6 shows carnation stems dipped in tamarind seed xyloglucan in theupper row, or not dipped in the lower row, following 1 day of incubationat room temperature.

FIG. 7A shows apple slices dipped in tamarind seed xyloglucan in theupper row, or not dipped in the lower row, after 2 days of incubation;FIG. 7B shows the same slices after 5 days of incubation.

FIG. 8 shows the effect of dipping discs of Granny Smith apples in 40 mMsodium citrate pH 5.5 containing 1 mg/ml tamarind seed xyloglucan withor without 1.1 μM Vigna angularis xyloglucan endotransglycosylase16(VaXET16) or 5 ml of deionized water after incubation under ambientconditions for 3, 4, and 7 days. FIG. 8A shows the apple slices after 3days of incubation. FIG. 8B shows the apple slices after 4 days ofincubation. FIG. 8C shows the apple slices after 7 days of incubation.

FIG. 9 shows quantitative analysis of apple slice images to determinethe extent to which xyloglucan and VaXET16 prevent apple oxidation. FIG.9A shows a pixel intensity histogram of apple slices not dipped after 4days of incubation. FIG. 9B shows a pixel intensity histogram of appleslices dipped in 40 mM sodium citrate pH 5.5 after 4 days of incubation.FIG. 9C shows a pixel intensity histogram of apple slices dipped inxyloglucan after 4 days of incubation. FIG. 9D shows a pixel intensityhistogram of apple slices dipped in xyloglucan and VaXET16 after 4 daysof incubation. FIG. 9E shows a plot of the mean intensity vs. time forthe variously treated apple slices.

FIG. 10A shows photographs of culture plates containing potato slicesdipped in the indicated solutions. Photographs are taken at 0, 2.5, 5,and 22 hours of incubation. FIG. 10B shows photographs of culture platescontaining avocado slices dipped in the indicated solutions. Photographswere taken at 0 and 70 hours of incubation.

FIG. 11A-11H shows a series of laser scanning confocal microscope imagesthat compare a fruit, flower, or vegetable incubated with Arabidopsisthaliana xyloglucan endotransglycosylase 14 (AtXET14) in 150 mM sodiumchloride-20 mM phosphate pH 7.2 to incubated with AtXET14 andfluorescein isothiocyanate-labeled xyloglucan (FITC-XG) in 150 mM sodiumchloride-20 mM phosphate pH 7.2 overnight at ambient temperature. FIG.11A shows a confocal image of a section of an apple slice incubated withAtXET14. FIG. 11B shows a confocal image of a section of an apple sliceincubated with AtXET14 and FITC-XG. FIG. 11C shows a confocal image of asection of a carnation stem incubated with AtXET14. FIG. 11D shows aconfocal image of a section of a carnation stem incubated with AtXET14and FITC-XG. FIG. 11E shows a confocal image of a section of a bananastem incubated with AtXET14. FIG. 11F shows a confocal image of asection of a banana stem incubated with AtXET14 and FITC-XG. FIG. 11Gshows a confocal image of a section of a squash stem incubated withAtXET14. FIG. 11H shows a confocal image of a section of a squash stemincubated with AtXET14 and FITC-XG.

DEFINITIONS

As used herein, the singular forms “a”, “an”, and “the” are intended toinclude the plural forms as well, unless the context clearly indicatesotherwise.

Agricultural crop: The term “agricultural crop” means any plant orproduct thereof that is harvested at some point in its growth stage,such as fruits, vegetables, perishable plants, flowers, grains and otherstaple crops, medicinal herbs and plants, nuts or seeds, crops grown forspinning cloth or fibers, and perishable foodstuffs derived directlytherefrom, for use or consumption by humans or animals.

Functionalized xyloglucan oligomer: The term “functionalized xyloglucanoligomer” means a short chain xyloglucan oligosaccharide, includingsingle or multiple repeating units of xyloglucan, which has beenmodified by incorporating a chemical group. The xyloglucan oligomer ispreferably 1 to 3 kDa in molecular weight, corresponding to 1 to 3repeating xyloglucan units. The chemical group may be a compound ofinterest or a reactive group such as an aldehyde group, an amino group,an aromatic group, a carboxyl group, a halogen group, a hydroxyl group,a ketone group, a nitrile group, a nitro group, a sulfhydryl group, or asulfonate group. The incorporated reactive groups can be derivatizedwith a compound of interest to provide a direct agricultural benefit orto coordinate metal cations and/or to bind other chemical entities thatinteract (e.g., covalently, hydrophobically, electrostatically, etc.)with the reactive groups. The derivatization can be performed directlyon a functionalized xyloglucan oligomer comprising a reactive group orafter the functionalized xyloglucan oligomer comprising a reactive groupis incorporated into polymeric xyloglucan. Alternatively, the xyloglucanoligomer can be functionalized by incorporating directly a compound byusing a reactive group contained in the compound, e.g., an aldehydegroup, an amino group, an aromatic group, a carboxyl group, a halogengroup, a hydroxyl group, a ketone group, a nitrile group, a nitro group,a sulfhydryl group, or a sulfonate group. The terms “functionalizedxyloglucan oligomer” and “functionalized xyloglucan oligomer comprisinga chemical group” are used interchangedly herein.

Polymeric xyloglucan: The term “polymeric xyloglucan” means short,intermediate or long chain xyloglucan oligosaccharide or polysaccharideencompassing more than one repeating unit of xyloglucan, e.g., multiplerepeating units of xyloglucan. Most optimally, polymeric xyloglucanencompasses xyloglucan of 50-200 kDa number average molecular weight,corresponding to 50-200 repeating units. A repeating motif of xyloglucanis composed of a backbone of four beta-(1-4)-D-glucopyranose residues,three of which have a single alpha-D-xylopyranose residue attached atO-6. Some of the xylose residues are beta-D-galactopyranosylated at O-2,and some of the galactose residues are alpha-L-fucopyranosylated at O-2.The term “xyloglucan” herein is understood to mean polymeric xyloglucan.

Polymeric xyloglucan functionalized with a chemical group: The term“polymeric xyloglucan functionalized with a chemical group” means apolymeric xyloglucan that has been modified by incorporating a chemicalgroup. The polymeric xyloglucan is short, intermediate or long chainxyloglucan oligosaccharide or polysaccharide encompassing more than onerepeating unit of xyloglucan, e.g., multiple repeating units ofxyloglucan. The polymeric xyloglucan encompasses xyloglucan of 50-200kDa number average molecular weight, corresponding to 50-200 repeatingunits. A repeating motif of xyloglucan is composed of a backbone of fourbeta-(1-4)-D-glucopyranose residues, three of which have a singlealpha-D-xylopyranose residue attached at O-6. The chemical group may bea compound of interest or a reactive group such as an aldehyde group, anamino group, an aromatic group, a carboxyl group, a halogen group, ahydroxyl group, a ketone group, a nitrile group, a nitro group, asulfhydryl group, or a sulfonate group. The chemical group can beincorporated into a polymeric xylogucan by reacting the polymericxyloglucan with a functionalized xyloglucan oligomer in the presence ofxyloglucan endotransglycosylase. The incorporated reactive groups canthen be derivatized with a compound of interest. The derivatization canbe performed directly on a functionalized polymeric xyloglucancomprising a reactive group or after a functionalized xyloglucanoligomer comprising a reactive group is incorporated into a polymericxyloglucan. Alternatively, the polymeric xyloglucan can befunctionalized by incorporating directly a compound by using a reactivegroup contained in the compound, e.g., an aldehyde group, an aminogroup, an aromatic group, a carboxyl group, a halogen group, a hydroxylgroup, a ketone group, a nitrile group, a nitro group, a sulfhydrylgroup, or a sulfonate group.

Xyloglucan endotransglycosylase: The term “xyloglucanendotransglycosylase” means a xyloglucan:xyloglucanxyloglucanotransferase (EC 2.4.1.207) that catalyzes cleavage of aβ-(1→4) bond in the backbone of a xyloglucan and transfers thexyloglucanyl segment on to 0-4 of the non-reducing terminal glucoseresidue of an acceptor, which can be a xyloglucan or an oligosaccharideof xyloglucan. Xyloglucan endotransglycosylases are also known asxyloglucan endotransglycosylase/hydrolases or endo-xyloglucantransferases. Some xylan endotransglycosylases can possess differentactivities including xyloglucan and mannan endotransglycosylaseactivities. For example, xylan endotransglycosylase from ripe papayafruit can use heteroxylans, such as wheat arabinoxylan, birchwoodglucuronoxylan, and others as donor molecules. These xylans canpotentially play a similar role as xyloglucan while being much cheaperin cost since they can be extracted, for example, from pulp mill spentliquors and/or future biomass biorefineries.

Xyloglucan endotransglycosylase activity can be assayed by those skilledin the art using any of the following methods. The reduction in theaverage molecular weight of a xyloglucan polymer when incubated with amolar excess of xyloglucan oligomer in the presence of xyloglucanendotransglycosylase can be determined via liquid chromatography (Sulovaet al., 2003, Plant Physiol. Biochem. 41: 431-437) or via ethanolprecipitation (Yaanaka et al., 2000, Food Hydrocolloids 14: 125-128)followed by gravimetric or cellulose-binding analysis (Fry et al., 1992,Biochem. J. 282: 821-828), or can be assessed colorimetrically byassociation with iodine under alkaline conditions (Sulova et al., 1995,Analytical Biochemistry229: 80-85). Incorporation of a functionalizedxyloglucan oligomer into a xyloglucan polymer by incubation of thefunctionalized oligomer with xyloglucan in the presence of xyloglucanendotransglycosylase can be assessed, e.g., by incubating a radiolabeledxyloglucan oligomer with xyloglucan and xyloglucan endotransglycosylase,followed by filter paper-binding and measurement of filter paperradioactivity, or incorporation of a fluorescently or opticallyfunctionalized xyloglucan oligomer can be assessed similarly, monitoringfluorescence or colorimetrically analyzing the filter paper.

Xyloglucan oligomer: The term “xyloglucan oligomer” means a short chainxyloglucan oligosaccharide, including single or multiple repeating unitsof xyloglucan. Most optimally, the xyloglucan oligomer will be 1 to 3kDa in molecular weight, corresponding to 1 to 3 repeating xyloglucanunits.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to methods for modifying an agriculturalcrop comprising treating the agricultural crop with a compositionselected from the group consisting of (a) a composition comprising axyloglucan endotransglycosylase, a polymeric xyloglucan, and afunctionalized xyloglucan oligomer comprising a chemical group; (b) acomposition comprising a xyloglucan endotransglycosylase, a polymericxyloglucan functionalized with a chemical group, and a functionalizedxyloglucan oligomer comprising a chemical group; (c) a compositioncomprising a xyloglucan endotransglycosylase, a polymeric xyloglucanfunctionalized with a chemical group, and a xyloglucan oligomer; (d) acomposition comprising a xyloglucan endotransglycosylase, a polymericxyloglucan, and a xyloglucan oligomer; (e) a composition comprising axyloglucan endotransglycosylase and a polymeric xyloglucanfunctionalized with a chemical group; (f) a composition comprising axyloglucan endotransglycosylase and a polymeric xyloglucan; (g) acomposition comprising a xyloglucan endotransglycosylase and afunctionalized xyloglucan oligomer comprising a chemical group; (h) acomposition comprising a xyloglucan endotransglycosylase and axyloglucan oligomer, and (i) a composition of (a), (b), (c), (d), (e),(f), (g), or (h) without a xyloglucan endotransglycosylase, wherein themodified agricultural crop possesses an improved property compared tothe unmodified agricultural crop.

The present invention also relates to modified agricultural cropsobtained by such methods.

The present invention also relates to modified agricultural cropscomprising (a) a polymeric xyloglucan and a functionalized xyloglucanoligomer comprising a chemical group; (b) a polymeric xyloglucanfunctionalized with a chemical group and a functionalized xyloglucanoligomer comprising a chemical group; (c) a polymeric xyloglucanfunctionalized with a chemical group and a xyloglucan oligomer; (d) apolymeric xyloglucan and a xyloglucan oligomer; (e) a polymericxyloglucan functionalized with a chemical group; (f) a polymericxyloglucan; (g) a functionalized xyloglucan oligomer comprising achemical group; or (h) a xyloglucan oligomer, wherein the modifiedagricultural crop possesses an improved property compared to theunmodified agricultural crop.

The present invention further relates to a composition selected from thegroup consisting of (a) a composition comprising a xyloglucanendotransglycosylase, a polymeric xyloglucan, and a functionalizedxyloglucan oligomer comprising a chemical group; (b) a compositioncomprising a xyloglucan endotransglycosylase, a polymeric xyloglucanfunctionalized with a chemical group, and a functionalized xyloglucanoligomer comprising a chemical group; (c) a composition comprising axyloglucan endotransglycosylase, a polymeric xyloglucan functionalizedwith a chemical group, and a xyloglucan oligomer; (d) a compositioncomprising a xyloglucan endotransglycosylase, a polymeric xyloglucan,and a xyloglucan oligomer; (e) a composition comprising a xyloglucanendotransglycosylase and a polymeric xyloglucan functionalized with achemical group; (f) a composition comprising a xyloglucanendotransglycosylase and a polymeric xyloglucan; (g) a compositioncomprising a xyloglucan endotransglycosylase and a functionalizedxyloglucan oligomer comprising a chemical group; (h) a compositioncomprising a xyloglucan endotransglycosylase and a xyloglucan oligomer,and (i) a composition of (a), (b), (c), (d), (e), (f), (g), or (h)without a xyloglucan endotransglycosylase.

In one embodiment, the composition comprises a xyloglucanendotransglycosylase, a polymeric xyloglucan, and a functionalizedxyloglucan oligomer comprising a chemical group. In another embodiment,the composition comprises a xyloglucan endotransglycosylase, a polymericxyloglucan functionalized with a chemical group, and a functionalizedxyloglucan oligomer comprising a chemical group. In another embodiment,the composition comprises a xyloglucan endotransglycosylase, a polymericxyloglucan functionalized with a chemical group, and a xyloglucanoligomer. In another embodiment, the composition comprises a xyloglucanendotransglycosylase, a polymeric xyloglucan, and a xyloglucan oligomer.In another embodiment, the composition comprises a xyloglucanendotransglycosylase and a polymeric xyloglucan functionalized with achemical group. In another embodiment, the composition comprises axyloglucan endotransglycosylase and a polymeric xyloglucan. In anotherembodiment, the composition comprises a xyloglucan endotransglycosylaseand a functionalized xyloglucan oligomer comprising a chemical group. Inanother embodiment, the composition comprises a xyloglucanendotransglycosylase and a xyloglucan oligomer.

In another embodiment, the composition comprises a polymeric xyloglucanand a functionalized xyloglucan oligomer comprising a chemical group. Inanother embodiment, the composition comprises a polymeric xyloglucanfunctionalized with a chemical group and a functionalized xyloglucanoligomer comprising a chemical group. In another embodiment, thecomposition comprises a polymeric xyloglucan functionalized with achemical group and a xyloglucan oligomer. In another embodiment, thecomposition comprises a polymeric xyloglucan and a xyloglucan oligomer.In another embodiment, the composition comprises a polymeric xyloglucanfunctionalized with a chemical group. In another embodiment, thecomposition comprises a polymeric xyloglucan. In another embodiment, thecomposition comprises a functionalized xyloglucan oligomer comprising achemical group. In another embodiment, the composition comprises axyloglucan oligomer.

The modification of an agricultural crop with a composition of thepresent invention can be conducted in any useful medium. In anembodiment, the medium is an aqueous medium. In another embodiment, themedium is a partially aqueous medium. In another aspect, the medium is aslurry. In another aspect, the medium is an aqueous slurry. In anotheraspect, the medium is a non-aqueous slurry. In another aspect, themedium is a partially aqueous slurry. In another aspect, the medium is awaxy suspension. In another aspect, the medium is an emulsion.

In one aspect, the agricultural crop is harvested. In another aspect,the agricultural crop is not harvested.

The methods of the present invention prevent qualitative andquantitative loss of agricultural crops and processed crops thereof.Once harvested, produce rapidly undergoes senescence, degrades inappearance, nutrition value, texture, firmness, and/or desirability. Ina related manner, once harvested, produce can be spoiled by microbialdegradation. Once the protective skins, peels, or rinds of produce arepierced, the produce is subject to oxidative damage, dehydration, lossof desirable appearance, and potential microbial degradation. Followingharvest, produce is shipped to wholesalers, dealers, and/or aggregators,and then to consumer markets. Sale must be necessarily expedited tominimize loss, thereby increasing associated costs. Treating cutflowers, fruits, vegetables or other agricultural crops with a solutionof polymeric xyloglucan, a naturally occurring plant polysaccharide, ormore preferably with a solution of polymeric xyloglucan and xyloglucanendotransglycosylase provides protection of the treated produce from,for example, degradation, spoilage, and the onset of negativeappearance. The polymeric xyloglucan can be functionalized with acompound, for example, with preservatives or hydrophobic chemicalmoieties, and the presence of xyloglucan endotransglycosylase permitssurface coating of the produce with the introduced functionalizationhaving, for example, the effect of preventing loss of water and keepingthe produce from drying out. Food-safe anti-microbial compounds, such asbacteriostatic or bacteriocidal compounds, can similarly be introducedin this manner.

In one aspect, the functionalization can provide any functionally usefulchemical moiety.

The xyloglucan endotransglycosylase is preferably present at about 0.1nM to about 1 mM, e.g., about 10 nM to about 100 μM or about 0.5 μM toabout 5 μM, in the composition.

The polymeric xyloglucan or polymeric xyloglucan functionalized with achemical group is preferably present at about 1 mg per g to about 1 gper g of the composition, e.g., about 10 mg to about 950 mg or about 100mg to about 900 mg per g of the composition.

When the xyloglucan oligomer or the functionalized xyloglucan oligomeris present without polymeric xyloglucan or polymeric xyloglucanfunctionalized with a chemical group, the xyloglucan oligomer or thefunctionalized xyloglucan oligomer is preferably present at about 1 mgto about 1 g per g of the composition, e.g., about 10 mg to about 950 mgor about 100 mg to about 900 mg per g of the composition.

When present with the polymeric xyloglucan or polymeric xyloglucanfunctionalized with a chemical group, the xyloglucan oligomer or thefunctionalized xyloglucan oligomer is preferably present with thepolymeric xyloglucan at about 50:1 to about 0.5:1 molar ratio ofxyloglucan oligomer or functionalized xyloglucan oligomer to polymericxyloglucan or polymeric xyloglucan functionalized with a chemical group,e.g., about 10:1 to about 1:1 or about 5:1 to about 1:1 molar ratio ofxyloglucan oligomer or functionalized xyloglucan oligomer to polymericxyloglucan or polymeric xyloglucan functionalized with a chemical group.

The polymeric xyloglucan or polymeric xyloglucan functionalized with achemical group is preferably present at about 1 ng to about 1 g per g ofthe agricultural crop, e.g., about 10 μg to about 100 mg or about 1 mgto about 50 mg per g of the agricultural crop.

When the xyloglucan oligomer or the functionalized xyloglucan oligomeris present without polymeric xyloglucan or polymeric xyloglucanfunctionalized with a chemical group, the xyloglucan oligomer or thefunctionalized xyloglucan oligomer is preferably present at about 1 ngper g to about 1 g per g of the agricultural crop, e.g., about 10 μg toabout 100 mg or about 1 mg to about 50 mg per g of the agriculturalcrop.

When present with the polymeric xyloglucan or polymeric xyloglucanfunctionalized with a chemical group, the xyloglucan oligomer or thefunctionalized xyloglucan oligomer is preferably present with thepolymeric xyloglucan at about 50:1 to about 0.5:1, e.g., about 10:1 toabout 1:1 or about 5:1 to about 1:1 molar ratio of xyloglucan oligomeror functionalized xyloglucan oligomer to polymeric xyloglucan orpolymeric xyloglucan functionalized with a chemical group.

The xyloglucan endotransglycosylase is preferably present at about 0.1nM to about 1 mM, e.g., about 10 nM to about 100 μM or about 0.5 μM toabout 5 μM.

The concentration of polymeric xyloglucan, polymeric xyloglucanfunctionalized with a chemical group, xyloglucan oligomer, orfunctionalized xyloglucan oligomer comprising a chemical groupincorporated onto or into the agricultural crop is about 1 pg to about500 mg per g of the agricultural crop, e.g. about 0.1 μg to about 50 mgor about 1 to about 5 mg per g of the agricultural crop.

Agricultural Crops

In the methods of the present invention, the agricultural crops can beany plant, or part thereof, grown for human or animal use orconsumption.

In one aspect, the agricultural crops are grown for human food. Inanother aspect, the agricultural crops are grown for silage. In anotheraspect, the agricultural crops are grown for animal or livestock feed.In another aspect, the agricultural crops are grown for seedlings,saplings, or transplant. In another aspect, the agricultural crops areornamental. In another aspect, the agricultural crops are trees. Inanother aspect, the agricultural crops are trees grown for timber. Inanother aspect, the agricultural crops are trees grown as Christmastrees. In another aspect, the agricultural crops are trees grown forfruit, vegetable, or nut production. In another aspect, the agriculturalcrops are bushes or shrubs. In another aspect, the agricultural cropsare grasses. In another aspect, the agricultural crops are flowers grownfor the cut flower market. In another aspect, the agricultural crops areflowers grown as houseplants. In another aspect, the agricultural cropsare grown for medicinal or homeopathic compounds. In another aspect, theagricultural crops are grown for fibers. In another aspect, theagricultural crop grown for fibers is cotton. In another aspect, theagricultural crop grown for fibers is hemp. In another aspect, theagricultural crop grown for fibers is flax. In another aspect, theagricultural crop grown for fibers is ramie. In another aspect, theagricultural crop grown for fibers is bamboo. In another aspect, theagricultural crops are grown for alcohol fermentation or beverageproduction. In another aspect, the agricultural crops are grown for drydistiller's grain.

In another aspect, the agricultural crop is a fruit. The fruit can beany type of fruit. The fruit can be apples, avocado, banana, berries,cucumbers, grape, tamarind, watermelon, cantaloupe, pumpkin, peach,plum, olive, orange, lemon, lime, pears, blackberry, pineapple, fig,mulberry, grains, sunflower, nuts, and non-botanical fruit. In oneaspect, the fruit are berries, (e.g., raspberries, blueberries, grapes,lingonberries, tomatoes, eggplant, cranberries, guava, pomegranate,chillies, and cucumbers). In another aspect, the fruit are pepo (e.g.,watermelon, cantaloupe, and pumpkin). In another aspect, the fruit aredrupe (e.g., peach, plum, and olive). In another aspect, the fruit arefollicles. In another aspect, the fruit are capsules (e.g., horsechestnut, cotton, and eucalyptus). In another aspect, the fruit arehesperidium (e.g., oranges, tangerines, grapefruits, lemons, and limes).In another aspect, the fruit are accessory fruit (e.g., apples andpears). In another aspect, the fruit are aggregate fruit (e.g.,blackberries, pineapples, and figs). In another aspect, the fruit aremultiple fruit (e.g., mulberries). In another aspect, the fruit areAchene (e.g., sunflower). In another aspect, the fruit are nuts (e.g.,walnut, oak, peanut, and almond). In another aspect, the fruit arenon-botanical fruit (e.g., juniper berries and rhubarb).

In another aspect, the agricultural crop is a vegetable. The vegetablecan be any edible plant or part thereof. The vegetables can beartichokes, asparagus, barley, bean sprouts, beans, black mustard,broccoli, Brussel sprouts, carrots, cauliflower, celery, clover, flax,garlic, ginger, hemp, India mustard, kale, kohlrabi, leek, lentil,lettuce, maize (corn), millet, oats, onion, pea, peanut, poppy,potatoes, radish, rhubarb, rice, rye, shallots, sorghum, soy, spinach,sweet potato, tamarind, triticale, watercress, or wheat.

In another aspect, the vegetable arises from the flower bud of a plant(e.g., broccoli, cauliflower, and artichokes). In another aspect, thevegetable arises from plant leaves (e.g., spinach, lettuce, kale, andwatercress). In another aspect, the vegetable arises from plant buds(e.g., Brussel sprouts). In another aspect, the vegetable arises fromplant shoots (e.g., asparagus and bean sprouts). In another aspect, thevegetable arises from plant stems (e.g., ginger and kohlrabi). Inanother aspect, the vegetable arises from plant tubers (e.g., potatoesand sweet potatoes). In another aspect, the vegetable arises from leafstems (e.g., celery and rhubarb). In another aspect, the vegetablearises from plant roots (e.g., carrots and radishes). In another aspect,the vegetable arises from plant bulbs (e.g., onions and shallots).

The vegetables can be leguminous vegetables, including the plants orseed of beans, soy, pea, lentil, clover, peanut, tamarind, and wisteria.

In another aspect, the agricultural crop is a grain. In another aspect,the grains are wheat, rice, oats, rye, triticale or barley. In anotheraspect, the grains are millet, sorghum, or maize (corn). In anotheraspect, the grains are mustards (e.g., black mustard and India mustard).In another aspect, the grains are grain legumes (e.g., peas, lentils,and beans). In another aspect, the grains are flax, hemp, or poppy.

In another aspect, the agricultural crop is a flower. The flower can beany flower. In one aspect, the flowers are field grown cut flowers. Inanother aspect, the flowers are greenhouse grown cut flowers. The flowercan be Ageratum houstonianum, Ammi majus, Antirrhinum majus,Callistephus chinensis, Celosia cristata, Centaurea cyanus, CentaureaAmericana, Clarkia amoena, Consolida regalis, Dianthus barbatus, Eustomagrandiflorum, Gypsophila elegans, Helianthus debilis cucumerifolius,Iberis amara, Limonium sinuatum, Nigella damascena, Scabiosaatropurpurea, Zinnia elegans, Achillea filipendulina, Artemisialudoviciana, Asclepias incarnate, Asclepias tuberosa, Aster novi-belgii,Aster ericoides, Astilbe, Chrysanthemum x superbum, Echinops bannaticus,Echinops exaltatus, Echinops ritro, Echinops sphaerocephalus, Eryngiumamethystinum, Eryngium planum, Eryngium alpinum, Gypsophila paniculata,Liatris, Paeonia, Platycodon grandiflorum, Salvia farinacea, Scabiosacaucasica, Solidago, Allium, Gladiolus, Lilium, Rosa, Antirrhinum,Gerbera, Tulipa, or Gladiolus.

In one aspect, the cut flowers are annuals. The annual flowers can beAgeratum houstonianum, Ammi majus, Antirrhinum majus, Callistephuschinensis, Celosia cristata, Centaurea cyanus, Centaurea Americana,Clarkia amoena, Consolida regalis, Dianthus barbatus, Eustomagrandiflorum, Gypsophila elegans, Helianthus debilis cucumerifolius,Iberis amara, Limonium sinuatum, Nigella damascena, Scabiosaatropurpurea, and Zinnia elegans.

In another aspect, the cut flowers are perennials. The perennial flowerscan be Achillea filipendulina, Artemisia ludoviciana, Asclepiasincarnate, Asclepias tuberosa, Aster novi-belgii, Aster ericoides,Astilbe, Chrysanthemum x superbum, Echinops bannaticus, Echinopsexaltatus, Echinops ritro, Echinops sphaerocephalus, Eryngiumamethystinum, Eryngium planum, Eryngium alpinum, Gypsophila paniculata,Liatris, Paeonia, Platycodon grandiflorum, Salvia farinacea, Scabiosacaucasica, and Solidago.

In another aspect, the cut flowers are bulbs. The flower bulbs can beAllium, Gladiolus, and Lilium. In another aspect, the cut flowers aretraditionally cut flowers such as chrysanthemums, carnations, and roses.In another aspect, the cut flowers are nontraditional cut flowers suchas lilies (Lilium), snapdragons (Antirrhinum), gerbera (Gerbera), tulips(Tulipa), and gladiolas (Gladiolus). In another aspect, the cut flowersare shipped from South America, Holland or the Caribbean. In anotheraspect, the cut flowers are shipped from local farms.

In another aspect, the agricultural crop is a spice. The spice can beAjwain (Trachyspermum ammi), Akudjura (Solanum centrale), Alexanders(Smyrnium olusatrum), Alkanet (Alkanna tinctoria), Alligator pepper,Mbongo spice (mbongochobi), Hepper pepper (Aframomum danielli, A.citratum, A. exscapum), Allspice (Pimenta dioica), Angelica (Angelicaarchangelica), Anise (Pimpinella anisum), Anise Hyssop (Agastachefoeniculum), Aniseed myrtle (Syzygium anisatum), Annatto (Bixaorellana), Apple mint (Mentha suaveolens, Mentha x rotundifolia andMentha x villosa), Artemisia (Artemisia spp.), Asafoetida (Ferulaassafoetida), Asarabacca (Asarum europaeum), Avens (Geum urbanum),Avocado leaf (Peresea americana), Barberry (Berberis vulgaris and otherBerberis spp.), Sweet basil (Ocimum basilicum), Lemon basil (Ocimum xcitriodorum), Thai basil (O. basilicum var. thyrsiflora), Holy Basil(Ocimum tenuiflorum), Bay leaf (Laurus nobilis), Bee balm (Monardadidyma), Boldo (Peumus boldus), Borage (Borago officinalis), Blackcardamom (Amomum subulatum, Amomum costatum), Black mustard (Brassicanigra), Blue fenugreek, Blue melilot (Trigonella caerulea), Brownmustard (Brassica juncea), Caraway (Carum carvi), White mustard (Sinapisalba), White cardamom (Elettaria cardamomum), Carob (Ceratonia siliqua),Catnip (Nepeta cataria), Cassia (Cinnamomum aromaticum), Cayenne pepper(Capsicum annuum), Celery leaf (Apium graveolens), Celery seed (Apiumgraveolens), Chervil (Anthriscus cerefolium), Chicory (Cichoriumintybus), Chili pepper (Capsicum spp.), Chives (Allium schoenoprasum),Sweet Cicely (Myrrhis odorata), Cilantro or coriander (Coriandrumsativum), Cinnamon, (Cinnamomum burmannii, Cinnamomum loureiroi,Cinnamomum verum, Cinnamomum zeylanicum), White Cinnamon (Canellawinterana), Myrtle Cinnamon (Backhousia myrtifolia), Clary sage (Salviasclarea), Clove (Syzygium aromaticum), Costmary (Tanacetum balsamita),Cuban oregano (Plectranthus amboinicus), Cubeb pepper (Piper cubeba),Cudweed (Gnaphalium spp.), Culantro, culangot or long coriander(Eryngium foetidum), Cumin (Cuminum cyminum), Curry leaf (Murrayakoenigii), Curry plant (Helichrysum italicum), Dill (Anethumgraveolens), Elderflower (Sambucus spp.), Epazote (Dysphaniaambrosioides), Fennel (Foeniculum vulgare), Fenugreek (Trigonellafoenum-graecum), File powder (Sassafras albidum), Fingerroot, Krachai orTemu kuntji (Boesenbergia rotunda), Greater Galangal (Alpinia galanga),Lesser Galangal (Alpinia officinarum), Galingale (Cyperus spp.), Garlicchives (Allium tuberosum), Garlic (Allium sativum), Elephant Garlic(Allium ampeloprasum var. ampeloprasum), Ginger (Zingiber officinale),Torch Ginger (Etlingera elatior), Golpar (Heracleum persicum), Grains ofparadise (Aframomum melegueta), Grains of Selim, Kani pepper (Xylopiaaethiopica), Horseradish (Armoracia rusticana), Houttuynia cordata,Huacatay, Mexican marigold, Mint marigold (Tagetes minuta), Hyssop(Hyssopus officinalis), Indonesian bay leaf, Daun salam (Syzygiumpolyanthum), Jasmine flowers (Jasminum spp.), Jimbu (Allium hypsistum),Juniper berry (Juniperus communis), Kaffir lime leaves (Citrus hystrix),Kala zeera (or kala jira), Black cumin (Bunium persicum), Kawakawa seeds(Macropiper excelsum), Kencur (Kaempferia galanga), Keluak (Pangiumedule), Vietnamese balm (Elsholtzia ciliata), Kokam seed (Garciniaindica), Korarima, Ethiopian cardamom (Aframomum corrorima), Koseretleaves (Lippia adoensis), Lavender (Lavandula spp.), Lemon balm (Melissaofficinalis), Lemongrass (Cymbopogon), Lemon ironbark (Eucalyptusstaigeriana), Lemon myrtle (Backhousia citriodora), Lemon verbena(Lippia citriodora), Leptotes bicolor, Lesser calamint (Calaminthanepeta), nipitella, Licorice (Glycyrrhiza glabra), Lime flower (Tiliaspp.), Lovage (Levisticum officinale), Mace (Myristica fragrans), St.Lucie cherry (Prunus mahaleb), Marjoram (Origanum majorana), Marshmallow (Althaea officinalis), Mastic (Pistacia lentiscus), Mint (Menthaspp.), Mountain horopito (Pseudowintera colorata), Musk mallow, abelmosk(Abelmoschus moschatus), Nasturtium (Tropaeolum majus), Nigella (Nigellasativa), Njangsa (Ricinodendron heudelotii), Nutmeg (Myristicafragrans), Neem, Olida (Eucalyptus olida), Oregano (Origanum), Orrisroot (Iris), Pandan flower, kewra (Pandanus odoratissimus), Pandan leaf(Pandanus amaryllifolius), Paprika (Capsicum annuum), Paracress(Spilanthes acmella, Soleracea), Parsley (Petroselinum crispum), Blackpepper, White pepper or Green pepper (Piper nigrum), Dorrigo pepper(Tasmannia stipitata), Long pepper (Piper longum) Mountain pepper(Tasmannia lanceolata), Peppermint (Mentha piperata), Peppermint gumleaf (Eucalyptus dives), Perilla (Perilla spp.), Peruvian pepper(Schinus molle), Pandanus amaryllifolius, Brazilian pepper (Schinusterebinthifolius), Quassia (Quassia amara), Ramsons (Allium ursinum),Rice paddy herb (Limnophila aromatica), Rosemary (Rosmarinusofficinalis), Rue (Ruta graveolens), Safflower (Carthamus tinctorius),Saffron (Crocus sativus), Sage (Salvia officinalis), Saigon cinnamon(Cinnamomum loureiroi), Salad burnet (Sanguisorba minor), Salep (Orchismascula), Sassafras (Sassafras albidum), Summer savory (Saturejahortensis), Winter savory (Satureja montana), Shiso (Perillafrutescens), Sorrel (Rumex acetosa), Sheep sorrel (Rumex acetosella),Spearmint (Mentha spicata), Spikenard (Nardostachys grandiflora or N.jatamansi), Star anise (Illicium verum), Sumac (Rhus coriaria), Sweetwoodruff (Galium odoratum), Szechuan pepper (Zanthoxylum piperitum),Tarragon (Artemisia dracunculus), Thyme (Thymus vulgaris), Lemon thyme(Thymus x citriodorus), Turmeric (Curcuma longa), Vanilla (Vanillaplanifolia), Vietnamese cinnamon (Cinnamomum loureiroi), Vietnamesecoriander (Persicaria odorata), Voatsiperifery (Piper borbonense),Wasabi (Wasabia japonica), Water-pepper (Polygonum hydropiper),Watercress (Rorippa nasturtium-aquatica), Wattleseed (Acacia), Wildbetel (Piper sarmentosum), Wild thyme (Thymus serpyllum), Willow herb(Epilobium parviflorum), Wintergreen (Gaultheria procumbens), Wood avens(Geum urbanum), Woodruff (Galium odoratum), absinthe (Artemisiaabsinthium), or Yerba buena.

The leaves, stems, stalks, shoots, seeds, roots, and/or fruit of anagricultural crop may be treated according to the methods of the presentinvention. The agricultural crop can be subsequently prepared fordisplay or consumption, either by cutting, slicing, peeling, deseeding,dehusking, or other methods known in the art.

Improved Properties

Treatment of an agricultural crop according to the methods of thepresent invention imparts an improved property to the agricultural crop,e.g., prior to harvest or post-harvest.

The improved property can be one or more improvements including, but notlimited to, reducing or preventing oxidative browning, dehydration,desiccation, bacterial, fungal, microbial, animal, or insect pestinfestation, senescence, early ripening, and softening. The one or moreimproved properties can also be physical improvements includingprevention of bruising, resistance to crushing, prevention orenhancement of clustering, and aggregation or association. The improvedproperty can also be one or more improvements including, but not limitedto, appearance, e.g., enhanced color or artificial coloration. The oneor more improved properties can also be resistance to adverseenvironmental factors, e.g., sun or UV damage. The improved property canalso be improved taste, e.g., by carbohydrate, salt or food additivefunctionalization.

In one aspect, the improved property is reducing or preventing oxidativebrowning. In another aspect, the improved property is reducing orpreventing dehydration. In another aspect, the improved property isreducing or preventing desiccation. In another aspect, the improvedproperty is reducing or preventing bacterial pest infestation. Inanother aspect, the improved property is reducing or preventing fungalpest infestation. In another aspect, the improved property is reducingor preventing microbial pest infestation. In another aspect, theimproved property is reducing or preventing animal pest infestation. Inanother aspect, the improved property is reducing or preventing insectpest infestation. In another aspect, the improved property is reducingor preventing senescence. In another aspect, the improved property isreducing or preventing early ripening. In another aspect, the improvedproperty is reducing or preventing softening. In another aspect, theimproved property is prevention of bruising, resistance to crushing,prevention or enhancement of clustering, and aggregation or association.In another aspect, the improved property is resistance to crushing. Inanother aspect, the improved property is prevention or enhancement ofclustering. In another aspect, the improved property is aggregation orassociation. In another aspect, the improved property is improvedappearance. In another aspect, the improved property is resistance toadverse environmental factors. In another aspect, the improved propertyis improved taste.

In one aspect, the improved property protects an agricultural crop priorto harvest. In another aspect, the improved property extends thetransportation time to market. In another aspect, the improved propertyextends the shelf-life of an agricultural crop. In another aspect, theimproved property increases nutritional value of an agricultural cropfor longer periods.

Polymeric Xyloglucan

In the methods of the present invention, the polymeric xyloglucan can beany xyloglucan. In one aspect, the polymeric xyloglucan is obtained fromnatural sources. In another aspect, the polymeric xyloglucan issynthesized from component carbohydrates, UDP- or GDP-carbohydrates, orhalogenated carbohydrates by any means used by those skilled in the art.In another aspect, the natural source of polymeric xyloglucan istamarind seed or tamarind kernel powder, nasturtium, or plants of thegenus Tropaeolum, particularly Tropaeolum majus. The natural source ofpolymeric xyloglucan may be seeds of various dicotyledonous plants suchas Hymenaea courbaril, Leguminosae-Caesalpinioideae including the generaCynometreae, Amherstieae, and Sclerolobieae. The natural source ofpolymeric xyloglucan may also be the seeds of plants of the familiesPrimulales, Annonaceae, Limnanthaceae, Melianthaceae, Pedaliaceae, andTropaeolaceae or subfamily Thunbergioideae. The natural source ofpolymeric xyloglucan may also be the seeds of plants of the familiesBalsaminaceae, Acanthaceae, Linaceae, Ranunculaceae, Sapindaceae, andSapotaceae or non-endospermic members of family Leguminosae subfamilyFaboideae. In another aspect, the natural source of polymeric xyloglucanis the primary cell walls of dicotyledonous plants. In another aspect,the natural source of polymeric xyloglucan may be the primary cell wallsof nongraminaceous, monocotyledonous plants.

The natural source polymeric xyloglucan may be extracted by extensiveboiling or hot water extraction, or by other methods known to thoseskilled in the art. In one aspect, the polymeric xyloglucan may besubsequently purified, for example, by precipitation in 80% ethanol. Inanother aspect, the polymeric xyloglucan is a crude or enrichedpreparation, for example, tamarind kernel powder. In another aspect, thesynthetic xyloglucan may be generated by automated carbohydratesynthesis (Seeberger, Chem. Commun, 2003, 1115-1121), or by means ofenzymatic polymerization, for example, using a glycosynthase (Spaduit etal., 2011, J. Am. Chem. Soc. 133: 10892-10900).

In one aspect, the average molecular weight of the polymeric xyloglucanranges from about 2 kDa to about 500 kDa, e.g., about 2 kDa to about 400kDa, about 3 kDa to about 300 kDa, about 3 kDa to about 200 kDa, about 5kDa to about 100 kDa, about 5 kDa to about 75 kDa, about 7.5 kDa toabout 50 kDa, or about 10 kDa to about 30 kDa. In another aspect, thenumber of repeating units is about 2 to about 500, e.g., about 2 toabout 400, about 3 to about 300, about 3 to about 200, about 5 to about100, about 7.5 to about 50, or about 10 to about 30. In another aspect,the repeating unit is any combination of G, X, L, F, S, T and Jsubunits, according to the nomenclature of Fry et al. (PhysiologiaPlantarum, 89: 1-3, 1993). In another aspect, the repeating unit iseither fucosylated or non-fucosylated XXXG-type polymeric xyloglucancommon to dicotyledons and nongraminaceous monocots. In another aspect,the polymeric xyloglucan is O-acetylated. In another aspect thepolymeric xyloglucan is not O-acetylated. In another aspect, side chainsof the polymeric xyloglucan may contain terminal fucosyl residues. Inanother aspect, side chains of the polymeric xyloglucan may containterminal arabinosyl residues. In another aspect, side chains of thepolymeric xyloglucan may contain terminal xylosyl residues.

For purposes of the present invention, references to the term xyloglucanherein refer to polymeric xyloglucan.

Xyloglucan Oligomer

In the methods of the present invention, the xyloglucan oligomer can beany xyloglucan oligomer. The xyloglucan oligomer may be obtained bydegradation or hydrolysis of polymeric xyloglucan from any source. Thexyloglucan oligomer may be obtained by enzymatic degradation ofpolymeric xyloglucan, e.g., by quantitative or partial digestion with axyloglucanase or endoglucanase (endo-β-1-4-glucanase). The xyloglucanoligomer may be synthesized from component carbohydrates, UDP- orGDP-carbohydrates, or halogenated carbohydrates by any of the mannerscommonly used by those skilled in the art.

In one aspect, the average molecular weight of the xyloglucan oligomerranges from 0.5 kDa to about 500 kDa, e.g., about 1 kDa to about 20 kDa,about 1 kDa to about 10 kDa, or about 1 kDa to about 3 kDa. In anotheraspect, the number of repeating units is about 1 to about 500, e.g.,about 1 to about 20, about 1 to about 10, or about 1 to about 3. In themethods of the present invention, the xyloglucan oligomer is optimallyas short as possible (i.e., 1 repeating unit, or about 1 kDa inmolecular weight) to maximize the solubility and solution molarity pergram of dissolved xyloglucan oligomer, while maintaining substratespecificity for xyloglucan endotransglycosylase activity. In anotheraspect, the xyloglucan oligomer comprises any combination of G (β-Dglucopyranosyl-), X (α-D-xylopyranosyl-(1→6)-β-D-glucopyranosyl-), L(β-D-galactopyranosyl-(1→2)-α-D-xylopyranosyl-(1→6)-β-D-glucopyranosyl-),F(α-L-fuco-pyranosyl-(1→2)-β-D-galactopyranosyl-(1→)-α-D-xylopyranosyl-(1→6)-β-D-glucopyranosyl-),S(α-L-arabinofurosyl-(1→2)-α-D-xylopyranosyl-(1→6)-β-D-glucopyranosyl-),T(α-L-arabino-furosyl-(1→3)-α-L-arabinofurosyl-(1→2)-α-D-xylopyranosyl-(1→6)-β-D-glucopyranosyl-),and J(α-L-galactopyranosyl-(1→2)-β-D-galactopyranosyl-(1→2)-α-D-xylopyranosyl-(1→6)-β-D-gluco-pyranosyl-)subunits according to the nomenclature of Fry et al. (PhysiologiaPlantarum 89: 1-3, 1993). In another aspect, the xyloglucan oligomer isthe XXXG heptasaccharide common to dicotyledons and nongraminaceousmonocots. In another aspect, the xyloglucan oligomer is O-acetylated. Inanother aspect, the xyloglucan oligomer is not O-acetylated. In anotheraspect, side chains of the xyloglucan oligomer may contain terminalfucosyl residues. In another aspect, side chains of the xyloglucanoligomer may contain terminal arabinosyl residues. In another aspect,side chains of the xyloglucan oligomer may contain terminal xylosylresidues.

Functionalization of Xyloglucan Oligomer and Polymeric Xyloglucan

The xyloglucan oligomer can be functionalized by incorporating anychemical group known to those skilled in the art. The chemical group maybe a compound of interest or a reactive group such as an aldehyde group,an amino group, an aromatic group, a carboxyl group, a halogen group, ahydroxyl group, a ketone group, a nitrile group, a nitro group, asulfhydryl group, or a sulfonate group.

In one aspect, the chemical group is an aldehyde group.

In another aspect, the chemical group is an amino group. The amino groupcan be incorporated into polymeric xyloglucan by reductive amination.Alternatively, the amino group can be an aliphatic amine or an aromaticamine (e.g., aniline). The aliphatic amine can be a primary, secondaryor tertiary amine. Primary, secondary, and tertiary amines are nitrogensbound to one, two and three carbons, respectively. In one aspect, theprimary amine is C₁-C₈, e.g., ethylamine. In another aspect, each carbonin the secondary amine is C₁-C₈, e.g., diethylamine. In another aspect,each carbon in the tertiary amine is C₁-C₈, e.g., triethyamine.

In another aspect, the chemical group is an aromatic group. The aromaticgroup can be an arene group, an aryl halide group, a phenolic group, aphenylamine group, a diazonium group, or a heterocyclic group.

In another aspect, the chemical group is a carboxyl group. The carboxylgroup can be an acyl halide, an amide, a carboxylic acid, an ester, or athioester.

In another aspect, the chemical group is a halogen group. The halogengroup can be fluorine, chlorine, bromine, or iodine.

In another aspect, the chemical group is a hydroxyl group.

In another aspect, the chemical group is a ketone group.

In another aspect, the chemical group is a nitrile group.

In another aspect, the chemical group is a nitro group.

In another aspect, the chemical group is a sulfhydryl group.

In another aspect, the chemical group is a sulfonate group.

The chemical reactive group can itself be the chemical group thatimparts a desired physical or chemical property to an agricultural crop.

By incorporation of chemical reactive groups in such a manner, oneskilled in the art can further derivatize the incorporated reactivegroups with compounds (e.g., macromolecules) that will impart a desiredphysical or chemical property to an agricultural crop. For example, theincorporated chemical group may react with the compound that imparts thedesired property to incorporate that group into the xyloglucan oligomervia a covalent bond. Alternatively, the chemical group may bind to thecompound that imparts the desired property in either a reversible orirreversible manner, and incorporate the compound via a non-covalentassociation. The derivatization can be performed directly on thefunctionalized xyloglucan oligomer or after the functionalizedxyloglucan oligomer is incorporated into polymeric xyloglucan.

Alternatively, the xyloglucan oligomer can be functionalized byincorporating directly a compound that imparts a desired physical orchemical property to a material by using a reactive group contained inthe compound or a reactive group incorporated into the compound, such asany of the groups described above.

On the other hand, the polymeric xyloglucan can be directlyfunctionalized by incorporating a reactive chemical group as describedabove. By incorporation of reactive chemical groups directly intopolymeric xyloglucan, one of skill in the art can further derivatize theincorporated reactive groups with compounds that will impart a desiredphysical or chemical property to a material. By incorporation of acompound directly into the polymeric xyloglucan, a desired physical orchemical property can also be directly imparted to a material.

In one aspect, the functionalization is performed by reacting thereducing end hydroxyl of the xyloglucan oligomer or the polymericxyloglucan. In another aspect, a non-reducing hydroxyl group, other thanthe non-reducing hydroxyl at position 4 of the terminal glucose, can bereacted. In another aspect, the reducing end hydroxyl and a non-reducinghydroxyl, other than the non-reducing hydroxyl at position 4 of theterminal glucose, can be reacted.

The chemical functional group can be added by enzymatic modification ofthe xyloglucan oligomer or polymeric xyloglucan, or by a non-enzymaticchemical reaction. In one aspect, enzymatic modification is used to addthe chemical functional group. In one embodiment of enzymaticmodification, the enzymatic functionalization is oxidation to a ketoneor carboxylate, e.g., by galactose oxidase. In another embodiment ofenzymatic modification, the enzymatic functionalization is oxidation toa ketone or carboxylate by AA9 Family oxidases (formerly glycohydrolaseFamily 61 enzymes).

In another aspect, the chemical functional group is added by anon-enzymatic chemical reaction. In one embodiment of the non-enzymaticchemical reaction, the reaction is incorporation of a reactive aminegroup by reductive amination of the reducing end of the carbohydrate asdescribed by Roy et al., 1984, Can. J. Chem. 62: 270-275, or Dalpathadoet al., 2005, Anal. Bioanal. Chem. 381: 1130-1137. In another embodimentof non-enzymatic chemical reaction, the reaction is incorporation of areactive ketone group by oxidation of the reducing end hydroxyl to aketone, e.g., by copper (II). In another embodiment of non-enzymaticchemical reaction, the reaction is oxidation of non-reducing endhydroxyl groups (e.g., of the non-glycosidic bonded position 6 hydroxylsof glucose or galactose) by (2,2,6,6-tetramethyl-piperidin-1-yl)oxyl(TEMPO), or the oxoammonium salt thereof, to generate an aldehyde orcarboxylic acid as described in Bragd et al., 2002, CarbohydratePolymers 49: 397-406, or Breton et al., 2007, Eur. J. Org. Chem. 10:1567-1570.

Xyloglucan oligomers or polymeric xyloglucan can be functionalized by achemical reaction with a compound containing more than one (i.e.bifunctional or multifunctional) chemical functional group comprising atleast one chemical functional group that is directly reactive withxyloglucan oligomer or polymeric xyloglucan. In one aspect, thebifunctional chemical group is a hydrocarbon containing a primary amineand a second chemical functional group. The second functional group canbe any of the other groups described above. In some aspects, the twofunctional groups are separated by hydrocarbon chains (linkers) ofvarious lengths as is well known in the art.

Xyloglucan oligomers or polymeric xyloglucan can be functionalized witha compound of interest by step-wise or concerted reaction wherein thexyloglucan oligomer or polymeric xyloglucan is functionalized asdescribed above, and the compound is reactive to the functionalizationintroduced therein. In one aspect of coupling via a functionalizedxyloglucan oligomer, an amino group is first incorporated into thexyloglucan oligomer by reductive amination and a reactive carbonyl issecondarily coupled to the introduced amino group. In another aspect ofcoupling via an amino-modified xyloglucan oligomer, the second couplingstep incorporates a chemical group, compound or macromolecule viacoupling an N-hydroxysuccinimidyl (NHS) ester or imidoester to theintroduced amino group. In a preferred embodiment, the NHS estersecondarily coupled to the introduced amino group is a component of amono or bi-functional crosslink reagent. In another aspect of couplingto a functionalized xyloglucan or xyloglucan oligomer, the firstreaction step comprises functionalization with a sulfhydryl group,either via reductive amination with an alkylthioamine (NH₂—(CH₂)_(n)—SH)at elevated temperatures in the presence of a reducing agent (Magid etal., 1996, J. Org. Chem. 61: 3849-3862), or via radical coupling (Wanget al., 2009, Arkivoc xiv: 171-180), followed by reaction of a maleimidegroup to the sulfhydryl. In some aspects, the reactive group in thecompound that imparts the desired property is separated from the rest ofthe compound by a hydrocarbon chain of an appropriate length, as is welldescribed in the art.

Non-limiting examples of compounds of interest that can be used tofunctionalize polymeric xyloglucan or xyloglucan oligomers, either bydirect reaction or via reaction with a xyloglucan-reactive compound,include peptides, polypeptides, proteins, hydrophobic groups,hydrophilic groups, flame retardants, dyes, color modifiers, specificaffinity tags, non-specific affinity tags, metals, metal oxides, metalsulfides, minerals, fungicides, herbicides, microbicides ormicrobiostatics, and non-covalent linker molecules.

In one aspect, the compound is a peptide. The peptide can be anantimicrobial peptide, a “self-peptide” designed to reduce allergenicityand immunogenicity, a cyclic peptide, glutathione, or a signalingpeptide (such as a tachykinin peptide, vasoactive intestinal peptide,pancreatic polypeptide related peptide, calcitonin peptide, lipopeptide,cyclic lipopeptide, or other peptide).

In another aspect, the compound is a polypeptide. The polypeptide can bea non-catalytically active protein (i.e., structural or bindingprotein), or a catalytically active protein (i.e., enzyme). Thepolypeptide can be an enzyme, an antibody, or an abzyme.

In another aspect, the compound is a compound comprising a hydrophobicgroup. The hydrophobic group can be polyurethane,polytetrafluoroethylene, or polyvinylidene fluoride.

In another aspect, the compound is a compound comprising a hydrophilicgroup. The hydrophilic group can be methacylate, methacrylamide, orpolyacrylate.

In another aspect, the compound is a flame retardant. Theflame-retardant can be aluminum hydroxide or magnesium hydroxide. Theflame-retardant can also be a compound comprising an organohalogen groupor an organophosphorous group.

In another aspect, the compound is a dye or pigment.

In another aspect, the compound is a specific affinity tag. The specificaffinity tag can be biotin, avidin, a chelating group, a crown ether, aheme group, a non-reactive substrate analog, an antibody, targetantigen, or a lectin.

In another aspect, the compound is a non-specific affinity tag. Thenon-specific affinity tag can be a polycation group, a polyanion group,a magnetic particle (e.g., magnetite), a hydrophobic group, an aliphaticgroup, a metal, a metal oxide, a metal sulfide, or a molecular sieve.

In another aspect, the compound is a fungicide. The fungicide can be acompound comprising a dicarboximide group (such as vinclozolin), aphenylpyrrole group (such as fludioxonil), a chlorophenyl group (such asquintozene), a chloronitrobenzene (such as dicloran), a triadiazolegroup (such as etridiazole), a dithiocarbamate group (such as mancozebor dimethyldithiocarbamate), or an inorganic molecule (such as copper orsulfur). In another aspect, the fungicide is a bacterium or bacterialspore such as Bacillus or a Bacillus spore.

In another aspect, the compound is a herbicide. The herbicide can beglyphosate, a synthetic plant hormone (such as a compound comprising a2,4-dichloropenoxyacetic acid group, a 2,4,5-trichlorophenoxyacetic acidgroup, a 2-methyl-4-chlorophenoxyacetic acid group, a2-(2-methyl-4-chlorophenoxy)propionic acid group, a2-(2,4-dichlorophenoxy)propionic acid group, or a(2,4-dichlorophenoxy)butyric acid group), or a compound comprising atriazine group (such as atrazine(2-chloro-4-(ethylamino)-6-isopropylamino)-s-triazine).

In another aspect, the compound is a bactericidal or bacteriostaticcompound. The bactericidal or bacteriostatic compound can be a copper orcopper alloy (such as brass, bronze, cupronickel, or copper-nickel-zincalloy), a sulfonamide group (such as sulfamethoxazole, sulfisomidine,sulfacetamide or sulfadiazine), a silver or organo-silver group, TiO₂,ZnO₂, an antimicrobial peptide, or chitosan.

In another aspect, the compound is a UV resistant compound. The UVresistant compound can be zinc or ZnO₂, kaolin, aluminum, aluminumoxides, or other UV-resistant compounds.

In another aspect, the compound is an anti-oxidant compound. Theanti-oxidant compound can be ascorbate, manganese, iodide, retinol, aterpenoid, tocopherol, a flavonoid or other anti-oxidant phenolic orpolyphenolic or other anti-oxidant compounds.

In another aspect, the compound is a non-covalent linker molecule.

In another aspect, the compound is a color modifier. The color modifiercan be a dye, fluorescent brightener, color modifier, or mordant (e.g.,alum, chrome alum).

Preparation of Modified Agricultural Crops

In the methods of the present invention, a modified agricultural cropcan be prepared by treating the agricultural crop with (a) a compositioncomprising a xyloglucan endotransglycosylase, a polymeric xyloglucan,and a functionalized xyloglucan oligomer comprising a chemical group;(b) a composition comprising a xyloglucan endotransglycosylase, apolymeric xyloglucan functionalized with a chemical group, and afunctionalized xyloglucan oligomer comprising a chemical group; (c) acomposition comprising a xyloglucan endotransglycosylase, a polymericxyloglucan functionalized with a chemical group, and a xyloglucanoligomer; (d) a composition comprising a xyloglucanendotransglycosylase, a polymeric xyloglucan, and a xyloglucan oligomer;(e) a composition comprising a xyloglucan endotransglycosylase and apolymeric xyloglucan functionalized with a chemical group; (f) acomposition comprising a xyloglucan endotransglycosylase and a polymericxyloglucan; (g) a composition comprising a xyloglucanendotransglycosylase and a functionalized xyloglucan oligomer comprisinga chemical group; (h) a composition comprising a xyloglucanendotransglycosylase and a xyloglucan oligomer, or (i) a composition of(a), (b), (c), (d), (e), (f), (g), or (h) without a xyloglucanendotransglycosylase, in a medium under conditions leading to a modifiedagricultural crop, wherein the modified agricultural crop possesses animproved property compared to the unmodified agricultural crop.

The methods are exemplified by, but are not limited to, improvedresistance to browning of apple and potato slices by treating theseagricultural crops, post harvest, with a solution comprising xyloglucanand xyloglucan endotransglycosylase. The xyloglucan can be anyxyloglucan, for example, tamarind kernel xyloglucan. The xyloglucanendotransglycosylase can be, for example, Vigna angularis XET16 orArabidopsis thaliana XET14. In the methods of the present invention,slices of fruit or vegetable (e.g., apple or potato) can be dipped in apH-controlled solution (e.g., a buffered solution such as sodiumcitrate) containing xyloglucan and xyloglucan endotransglycosylase. ThepH of the buffered solution can be between about 3 and about 9, e.g.,about 4 to about 8 or about 5 to about 7. The concentration of sodiumcitrate can be about 1 mM to about 1 M, e.g., about 5 mM to about 500mM, about 10 mM to about 100 mM, or about 20 mM to about 50 mM. Theconcentration of xyloglucan can be about 10 mg/L to about 100 g/L, e.g.,about 100 mg/L to about 10 g/L, about 500 mg/L to about 5 g/L, or about1 g/L to about 2 g/L. The concentration of xyloglucanendotransglycosylase can be about 1 nM to about 1 mM, e.g. about 10 nMto about 100 μM, about 100 nM to about 10 μM, or about 500 nM to about1.5 μM. The time length of the dip can be instantaneous to about 12hours, e.g., about 1 second to about 3 hours, about 10 seconds to about30 minutes, or about 30 seconds to about 2 minutes. The time length ofthe dip can be optimized to maximize the improved property, or can beoptimized to the method by one skilled in the art. The excess solutioncan be removed, for instance, by washing in water, by dipping in apH-controlled solution not containing xyloglucan, xyloglucanendotransglycosylase, or both, or by touching the slice of apple orpotato to the side of the container or to a paper towel or wipe.Alternatively, the excess solution can be left on the agricultural cropor left on the crop for an appropriate length of time prior to washing.In the current example, the excess solution is removed by touching thefruit or vegetable to the side of the container. In one aspect, thexyloglucan and xyloglucan endotransglycosylase can be separated into 2solutions and the agricultural crop dipped into each independently andsequentially. In another aspect, xyloglucan oligomers or functionalizedxyloglucan oligomers are added to the solution of xyloglucan andxyloglucan endotransglycosylase, or to one or the other or bothsolutions if the two components are separated into 2 solutions. Forexample, xyloglucan oligomers can be added to a solution of xyloglucanand xyloglucan endotransglycosylase at a molar ratio to xyloglucan ofabout 10⁻⁴ to about 100, e.g., about 10⁻³ to about 10 or about 10⁻² toabout 1. In this manner, one of skill in the art can use thetransglycosylase activity of xyloglucan endotransglycosylase to optimizethe size of the xyloglucan polymer and/or the degree offunctionalization of the xyloglucan to affect the optimal improvedproperty.

Sources of Xyloglucan Endotransglycosylases

Any xyloglucan endotransglycosylase that possesses suitable enzymeactivity at a pH and temperature appropriate for the methods of thepresent invention may be used. It is preferable that the xyloglucanendotransglycosylase is active over a broad pH and temperature range. Inan embodiment, the xyloglucan endotransglycosylase has a pH optimum inthe range of about 3 to about 10. In another embodiment, the xyloglucanendotransglycosylase has a pH optimum in the range of about 4.5 to about8.5. In another embodiment, the xyloglucan endotransglycosylase has acold denaturation temperature less than or equal to about 5° C. or amelting temperature of about 100° C. or higher. In another embodiment,the xyloglucan endotransglycosylase has a cold denaturation temperatureof less than or equal to 20° C. or a melting temperature greater than orequal to about 75° C.

The source of the xyloglucan endotransglycosylase used is not criticalin the present invention. Accordingly, the xyloglucanendotransglycosylase may be obtained from any source such as a plant,microorganism, or animal.

In one embodiment, the xyloglucan endotransglycosylase is obtained froma plant source. Xyloglucan endotransglycosylase can be obtained fromcotyledons of the family Fabaceae (synonyms: Leguminosae andPapilionaceae), preferably genus Phaseolus, in particular, Phaseolusaureus. Preferred monocotyledons are non-graminaceous monocotyledons andliliaceous monocotyledons. Xyloglucan endotransglycosylase can also beextracted from moss and liverwort, as described in Fry et al., 1992,Biochem. J. 282: 821-828. For example, the xyloglucanendotransglycosylase may be obtained from cotyledons, i.e., adicotyledon or a monocotyledon, in particular a dicotyledon selectedfrom the group consisting of azuki beans, canola, cauliflowers, cotton,poplar or hybrid aspen, potatoes, rapes, soy beans, sunflowers,thalecress, tobacco, and tomatoes, or a monocotyledon selected from thegroup consisting of wheat, rice, corn, and sugar cane. See, for example,WO 2003/033813 and WO 97/23683.

In another embodiment, the xyloglucan endotransglycosylase is obtainedfrom Arabidopsis thaliana (GENESEQP:AOE11231, GENESEQP:AOE93420,GENESEQP: BAL03414, GENESEQP:BAL03622, or GENESEQP:AWK95154); Caricapapaya (GENESEQP:AZR75725); Cucumis sativus (GENESEQP:AZV66490); Daucuscarota (GENESEQP:AZV66139); Festuca pratensis (GENESEQP:AZR80321);Glycine max (GENESEQP:AWK95154 or GENESEQP:AYF92062); Hordeum vulgare(GENESEQP:AZR85056, GENESEQP:AQY12558, GENESEQP:AQY12559, orGENESEQP:AWK95180); Lycopersicon esculentum (GENESEQP:ATZ45232);Medicago truncatula (GENESEQP:ATZ48025); Oryza sativa(GENESEQP:ATZ42485, GENESEQP:ATZ57524, or GENESEQP:AZR76430); Populustremula (GENESEQP:AWK95036); Sagittaria pygmaea (GENESEQP:AZV66468);Sorghum bicolor (GENESEQP:BAO79623 or GENESEQP:BAO79007); Vignaangularis (GENESEQP:ATZ61320); or Zea mays (GENESEQP:AWK94916).

In another embodiment, the xyloglucan endotransglycosylase is axyloglucan endotransglucosylase/hydrolase (XTH) with both hydrolytic andtransglycosylating activities. In a preferred embodiment, the ratio oftransglycosylation to hydrolytic rates is at least 10⁻² to 10⁷, e.g.,10⁻¹ to 10⁶ or 10 to 1000.

Production of Xyloglucan Endotransglycosylases

Xyloglucan endotransglycosylase may be extracted from plants. Suitablemethods for extracting xyloglucan endotransglycosylase from plants aredescribed Fry et al., 1992, Biochem. J. 282: 821-828; Sulova et al.,1998, Biochem. J. 330: 1475-1480; Sulova et al., 1995, Anal. Biochem.229: 80-85; WO 95/13384; WO 97/23683; or EP 562 836.

Xyloglucan endotransglycosylase may also be produced by cultivation of atransformed host organism containing the appropriate genetic informationfrom a plant, microorganism, or animal. Transformants can be preparedand cultivated by methods known in the art.

Techniques used to isolate or clone a gene are known in the art andinclude isolation from genomic DNA or cDNA, or a combination thereof.The cloning of the gene from genomic DNA can be effected, e.g., by usingthe polymerase chain reaction (PCR) or antibody screening of expressionlibraries to detect cloned DNA fragments with shared structuralfeatures. See, e.g., Innis et al., 1990, PCR: A Guide to Methods andApplication, Academic Press, New York. Other nucleic acid amplificationprocedures such as ligase chain reaction (LCR), ligation activatedtranscription (LAT) and polynucleotide-based amplification (NASBA) maybe used.

A nucleic acid construct can be constructed to comprise a gene encodinga xyloglucan endotransglycosylase operably linked to one or more controlsequences that direct the expression of the coding sequence in asuitable host cell under conditions compatible with the controlsequences. The gene may be manipulated in a variety of ways to providefor expression of the xyloglucan endotransglycosylase. Manipulation ofthe gene prior to its insertion into a vector may be desirable ornecessary depending on the expression vector. Techniques for modifyingpolynucleotides utilizing recombinant DNA methods are well known in theart.

The control sequence may be a promoter, a polynucleotide that isrecognized by a host cell for expression of a polynucleotide encoding axyloglucan endotransglycosylase. The promoter contains transcriptionalcontrol sequences that mediate the expression of the xyloglucanendotransglycosylase. The promoter may be any polynucleotide that showstranscriptional activity in the host cell including mutant, truncated,and hybrid promoters, and may be obtained from genes encodingextracellular or intracellular polypeptides either homologous orheterologous to the host cell.

Examples of suitable promoters for directing transcription of thenucleic acid constructs in a bacterial host cell are the promotersobtained from the Bacillus amyloliquefaciens alpha-amylase gene (amyQ),Bacillus licheniformis alpha-amylase gene (amyL), Bacillus licheniformispenicillinase gene (penP), Bacillus stearothermophilus maltogenicamylase gene (amyM), Bacillus subtilis levansucrase gene (sacB),Bacillus subtilis xylA and xylB genes, Bacillus thuringiensis cryIIIAgene (Agaisse and Lereclus, 1994, Molecular Microbiology 13: 97-107), E.coli lac operon, E. coli trc promoter (Egon et al., 1988, Gene 69:301-315), Streptomyces coelicolor agarase gene (dagA), and prokaryoticbeta-lactamase gene (Villa-Kamaroff et al., 1978, Proc. Natl. Acad. Sci.USA 75: 3727-3731), as well as the tac promoter (DeBoer et al., 1983,Proc. Natl. Acad. Sci. USA 80: 21-25). Further promoters are describedin “Useful proteins from recombinant bacteria” in Gilbert et al., 1980,Scientific American 242: 74-94; and in Sambrook et al., 1989, supra.Examples of tandem promoters are disclosed in WO 99/43835.

Examples of suitable promoters for directing transcription of thenucleic acid constructs in a filamentous fungal host cell are promotersobtained from the genes for Aspergillus nidulans acetamidase,Aspergillus niger neutral alpha-amylase, Aspergillus niger acid stablealpha-amylase, Aspergillus niger or Aspergillus awamori glucoamylase(glaA), Aspergillus oryzae TAKA amylase, Aspergillus oryzae alkalineprotease, Aspergillus oryzae triose phosphate isomerase, Fusariumoxysporum trypsin-like protease (WO 96/00787), Fusarium venenatumamyloglucosidase (WO 00/56900), Fusarium venenatum Daria (WO 00/56900),Fusarium venenatum Quinn (WO 00/56900), Rhizomucor miehei lipase,Rhizomucor miehei aspartic proteinase, Trichoderma reeseibeta-glucosidase, Trichoderma reesei cellobiohydrolase I, Trichodermareesei cellobiohydrolase II, Trichoderma reesei endoglucanase I,Trichoderma reesei endoglucanase II, Trichoderma reesei endoglucanaseIII, Trichoderma reesei endoglucanase V, Trichoderma reesei xylanase I,Trichoderma reesei xylanase II, Trichoderma reesei xylanase Ill,Trichoderma reesei beta-xylosidase, and Trichoderma reesei translationelongation factor, as well as the NA2-tpi promoter (a modified promoterfrom an Aspergillus neutral alpha-amylase gene in which the untranslatedleader has been replaced by an untranslated leader from an Aspergillustriose phosphate isomerase gene; non-limiting examples include modifiedpromoters from an Aspergillus niger neutral alpha-amylase gene in whichthe untranslated leader has been replaced by an untranslated leader froman Aspergillus nidulans or Aspergillus oryzae triose phosphate isomerasegene); and mutant, truncated, and hybrid promoters thereof. Otherpromoters are described in U.S. Pat. No. 6,011,147.

In a yeast host, useful promoters are obtained from the genes forSaccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiaegalactokinase (GAL1), Saccharomyces cerevisiae alcoholdehydrogenase/glyceraldehyde-3-phosphate dehydrogenase (ADH1, ADH2/GAP),Saccharomyces cerevisiae triose phosphate isomerase (TPI), Saccharomycescerevisiae metallothionein (CUP1), and Saccharomyces cerevisiae3-phosphoglycerate kinase. Other useful promoters for yeast host cellsare described by Romanos et al., 1992, Yeast 8: 423-488.

The control sequence may also be a transcription terminator, which isrecognized by a host cell to terminate transcription. The terminator isoperably linked to the 3′-terminus of the polynucleotide encoding thexyloglucan endotransglycosylase. Any terminator that is functional inthe host cell may be used in the present invention.

Preferred terminators for bacterial host cells are obtained from thegenes for Bacillus clausii alkaline protease (aprH), Bacilluslicheniformis alpha-amylase (amyL), and Escherichia coli ribosomal RNA(rrnB).

Preferred terminators for filamentous fungal host cells are obtainedfrom the genes for Aspergillus nidulans acetamidase, Aspergillusnidulans anthranilate synthase, Aspergillus niger glucoamylase,Aspergillus niger alpha-glucosidase, Aspergillus oryzae TAKA amylase,Fusarium oxysporum trypsin-like protease, Trichoderma reeseibeta-glucosidase, Trichoderma reesei cellobiohydrolase I, Trichodermareesei cellobiohydrolase II, Trichoderma reesei endoglucanase I,Trichoderma reesei endoglucanase II, Trichoderma reesei endoglucanaseIII, Trichoderma reesei endoglucanase V, Trichoderma reesei xylanase I,Trichoderma reesei xylanase II, Trichoderma reesei xylanase III,Trichoderma reesei beta-xylosidase, and Trichoderma reesei translationelongation factor.

Preferred terminators for yeast host cells are obtained from the genesfor Saccharomyces cerevisiae enolase, Saccharomyces cerevisiaecytochrome C (CYC1), and Saccharomyces cerevisiaeglyceraldehyde-3-phosphate dehydrogenase. Other useful terminators foryeast host cells are described by Romanos et al., 1992, supra.

The control sequence may also be an mRNA stabilizer region downstream ofa promoter and upstream of the coding sequence of a gene which increasesexpression of the gene.

Examples of suitable mRNA stabilizer regions are obtained from aBacillus thuringiensis cryIIIA gene (WO 94/25612) and a Bacillussubtilis SP82 gene (Hue et al., 1995, Journal of Bacteriology 177:3465-3471).

The control sequence may also be a leader, a nontranslated region of anmRNA that is important for translation by the host cell. The leader isoperably linked to the 5′-terminus of the polynucleotide encoding thexyloglucan endotransglycosylase. Any leader that is functional in thehost cell may be used.

Preferred leaders for filamentous fungal host cells are obtained fromthe genes for Aspergillus oryzae TAKA amylase and Aspergillus nidulanstriose phosphate isomerase.

Suitable leaders for yeast host cells are obtained from the genes forSaccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiae3-phosphoglycerate kinase, Saccharomyces cerevisiae alpha-factor, andSaccharomyces cerevisiae alcoholdehydrogenase/glyceraldehyde-3-phosphate dehydrogenase (ADH2/GAP).

The control sequence may also be a polyadenylation sequence, a sequenceoperably linked to the 3′-terminus of the polynucleotide and, whentranscribed, is recognized by the host cell as a signal to addpolyadenosine residues to transcribed mRNA. Any polyadenylation sequencethat is functional in the host cell may be used.

Preferred polyadenylation sequences for filamentous fungal host cellsare obtained from the genes for Aspergillus nidulans anthranilatesynthase, Aspergillus nigerglucoamylase, Aspergillus nigeralpha-glucosidase Aspergillus oryzae TAKA amylase, and Fusariumoxysporum trypsin-like protease.

Useful polyadenylation sequences for yeast host cells are described byGuo and Sherman, 1995, Mol. Cellular Biol. 15: 5983-5990.

The control sequence may also be a signal peptide coding region thatencodes a signal peptide linked to the N-terminus of a xyloglucanendotransglycosylase and directs the polypeptide into the cell'ssecretory pathway. The 5′-end of the coding sequence of thepolynucleotide may inherently contain a signal peptide coding sequencenaturally linked in translation reading frame with the segment of thecoding sequence that encodes the polypeptide. Alternatively, the 5′-endof the coding sequence may contain a signal peptide coding sequence thatis foreign to the coding sequence. A foreign signal peptide codingsequence may be required where the coding sequence does not naturallycontain a signal peptide coding sequence. Alternatively, a foreignsignal peptide coding sequence may simply replace the natural signalpeptide coding sequence in order to enhance secretion of thepolypeptide. However, any signal peptide coding sequence that directsthe expressed polypeptide into the secretory pathway of a host cell maybe used.

Effective signal peptide coding sequences for bacterial host cells arethe signal peptide coding sequences obtained from the genes for BacillusNCIB 11837 maltogenic amylase, Bacillus licheniformis subtilisin,Bacillus licheniformis beta-lactamase, Bacillus stearothermophilusalpha-amylase, Bacillus stearothermophilus neutral proteases (nprT,nprS, nprM), and Bacillus subtilis prsA. Further signal peptides aredescribed by Simonen and Palva, 1993, Microbiological Reviews 57:109-137.

Effective signal peptide coding sequences for filamentous fungal hostcells are the signal peptide coding sequences obtained from the genesfor Aspergillus niger neutral amylase, Aspergillus niger glucoamylase,Aspergillus oryzae TAKA amylase, Humicola insolens cellulase, Humicolainsolens endoglucanase V, Humicola lanuginosa lipase, and Rhizomucormiehei aspartic proteinase.

Useful signal peptides for yeast host cells are obtained from the genesfor Saccharomyces cerevisiae alpha-factor and Saccharomyces cerevisiaeinvertase. Other useful signal peptide coding sequences are described byRomanos et al., 1992, supra.

The control sequence may also be a propeptide coding sequence thatencodes a propeptide positioned at the N-terminus of a xyloglucanendotransglycosylase. The resultant polypeptide is known as a proenzymeor propolypeptide (or a zymogen in some cases). A propolypeptide isgenerally inactive and can be converted to an active polypeptide bycatalytic or autocatalytic cleavage of the propeptide from thepropolypeptide. The propeptide coding sequence may be obtained from thegenes for Bacillus subtilis alkaline protease (aprE), Bacillus subtilisneutral protease (nprT), Myceliophthora thermophila laccase (WO95/33836), Rhizomucor miehei aspartic proteinase, and Saccharomycescerevisiae alpha-factor.

Where both signal peptide and propeptide sequences are present, thepropeptide sequence is positioned next to the N-terminus of a xyloglucanendotransglycosylase and the signal peptide sequence is positioned nextto the N-terminus of the propeptide sequence.

The various nucleotide and control sequences may be joined together toproduce a recombinant expression vector that may include one or moreconvenient restriction sites to allow for insertion or substitution ofthe polynucleotide encoding the xyloglucan endotransglycosylase at suchsites. Alternatively, the polynucleotide may be expressed by insertingthe polynucleotide or a nucleic acid construct comprising thepolynucleotide into an appropriate vector for expression. In creatingthe expression vector, the coding sequence is located in the vector sothat the coding sequence is operably linked with the appropriate controlsequences for expression.

The recombinant expression vector may be any vector (e.g., a plasmid orvirus) that can be conveniently subjected to recombinant DNA proceduresand can bring about expression of the polynucleotide. The choice of thevector will typically depend on the compatibility of the vector with thehost cell into which the vector is to be introduced. The vector may be alinear or closed circular plasmid.

The vector may be an autonomously replicating vector, i.e., a vectorthat exists as an extrachromosomal entity, the replication of which isindependent of chromosomal replication, e.g., a plasmid, anextrachromosomal element, a minichromosome, or an artificial chromosome.The vector may contain any means for assuring self-replication.Alternatively, the vector may be one that, when introduced into the hostcell, is integrated into the genome and replicated together with thechromosome(s) into which it has been integrated. Furthermore, a singlevector or plasmid or two or more vectors or plasmids that togethercontain the total DNA to be introduced into the genome of the host cell,or a transposon, may be used.

The vector preferably contains one or more selectable markers thatpermit easy selection of transformed, transfected, transduced, or thelike cells. A selectable marker is a gene the product of which providesfor biocide or viral resistance, resistance to heavy metals, prototrophyto auxotrophs, and the like.

Examples of bacterial selectable markers are Bacillus licheniformis orBacillus subtilis dal genes, or markers that confer antibioticresistance such as ampicillin, chloramphenicol, kanamycin, neomycin,spectinomycin, or tetracycline resistance. Suitable markers for yeasthost cells include, but are not limited to, ADE2, HIS3, LEU2, LYS2,MET3, TRP1, and URA3. Selectable markers for use in a filamentous fungalhost cell include, but are not limited to, adeA(phosphoribosylaminoimidazole-succinocarboxamide synthase), adeB(phosphoribosylaminoimidazole synthase), amdS (acetamidase), argB(ornithine carbamoyltransferase), bar (phosphinothricinacetyltransferase), hph (hygromycin phosphotransferase), niaD (nitratereductase), pyrG (orotidine-5′-phosphate decarboxylase), sC (sulfateadenyltransferase), and trpC (anthranilate synthase), as well asequivalents thereof. Preferred for use in an Aspergillus cell areAspergillus nidulans or Aspergillus oryzae amdS and pyrG genes and aStreptomyces hygroscopicus bar gene. Preferred for use in a Trichodermacell are adeA, adeB, amdS, hph, and pyrG genes.

The selectable marker may be a dual selectable marker system asdescribed in WO 2010/039889. In one aspect, the dual selectable markeris an hph-tk dual selectable marker system.

The vector preferably contains an element(s) that permits integration ofthe vector into the host cell's genome or autonomous replication of thevector in the cell independent of the genome.

For integration into the host cell genome, the vector may rely on thepolynucleotide's sequence encoding the xyloglucan endotransglycosylaseor any other element of the vector for integration into the genome byhomologous or non-homologous recombination. Alternatively, the vectormay contain additional polynucleotides for directing integration byhomologous recombination into the genome of the host cell at a preciselocation(s) in the chromosome(s). To increase the likelihood ofintegration at a precise location, the integrational elements shouldcontain a sufficient number of nucleic acids, such as 100 to 10,000 basepairs, 400 to 10,000 base pairs, and 800 to 10,000 base pairs, whichhave a high degree of sequence identity to the corresponding targetsequence to enhance the probability of homologous recombination. Theintegrational elements may be any sequence that is homologous with thetarget sequence in the genome of the host cell. Furthermore, theintegrational elements may be non-encoding or encoding polynucleotides.On the other hand, the vector may be integrated into the genome of thehost cell by non-homologous recombination.

For autonomous replication, the vector may further comprise an origin ofreplication enabling the vector to replicate autonomously in the hostcell in question. The origin of replication may be any plasmidreplicator mediating autonomous replication that functions in a cell.The term “origin of replication” or “plasmid replicator” means apolynucleotide that enables a plasmid or vector to replicate in vivo.

Examples of bacterial origins of replication are the origins ofreplication of plasmids pBR322, pUC19, pACYC177, and pACYC184 permittingreplication in E. coli, and pUB110, pE194, pTA1060, and pAMβ1 permittingreplication in Bacillus.

Examples of origins of replication for use in a yeast host cell are the2 micron origin of replication, ARS1, ARS4, the combination of ARS1 andCEN3, and the combination of ARS4 and CEN6.

Examples of origins of replication useful in a filamentous fungal cellare AMA1 and ANSI (Gems et al., 1991, Gene 98: 61-67; Cullen et al.,1987, Nucleic Acids Res. 15: 9163-9175; WO 00/24883). Isolation of theAMA1 gene and construction of plasmids or vectors comprising the genecan be accomplished according to the methods disclosed in WO 00/24883.

More than one copy of a polynucleotide may be inserted into a host cellto increase production of a xyloglucan endotransglycosylase. An increasein the copy number of the polynucleotide can be obtained by integratingat least one additional copy of the sequence into the host cell genomeor by including an amplifiable selectable marker gene with thepolynucleotide where cells containing amplified copies of the selectablemarker gene, and thereby additional copies of the polynucleotide, can beselected for by cultivating the cells in the presence of the appropriateselectable agent.

The procedures used to ligate the elements described above to constructthe recombinant expression vectors are well known to one skilled in theart (see, e.g., Sambrook et al., 1989, supra).

The host cell may be any cell useful in the recombinant production of axyloglucan endotransglycosylase, e.g., a prokaryote or a eukaryote.

The prokaryotic host cell may be any Gram-positive or Gram-negativebacterium. Gram-positive bacteria include, but are not limited to,Bacillus, Clostridium, Enterococcus, Geobacillus, Lactobacillus,Lactococcus, Oceanobacillus, Staphylococcus, Streptococcus, andStreptomyces. Gram-negative bacteria include, but are not limited to,Campylobacter, E. coli, Flavobacterium, Fusobacterium, Helicobacter,Ilyobacter, Neisseria, Pseudomonas, Salmonella, and Ureaplasma.

The bacterial host cell may be any Bacillus cell including, but notlimited to, Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillusbrevis, Bacillus circulans, Bacillus clausii, Bacillus coagulans,Bacillus firmus, Bacillus lautus, Bacillus lentus, Bacilluslicheniformis, Bacillus megaterium, Bacillus pumilus, Bacillusstearothermophilus, Bacillus subtilis, and Bacillus thuringiensis cells.

The bacterial host cell may also be any Streptomyces cell including, butnot limited to, Streptomyces achromogenes, Streptomyces avermitilis,Streptomyces coelicolor, Streptomyces griseus, and Streptomyces lividanscells.

The introduction of DNA into a Bacillus cell may be effected byprotoplast transformation (see, e.g., Chang and Cohen, 1979, Mol. Gen.Genet. 168: 111-115), competent cell transformation (see, e.g., Youngand Spizizen, 1961, J. Bacteriol. 81: 823-829, or Dubnau andDavidoff-Abelson, 1971, J. Mol. Biol. 56: 209-221), electroporation(see, e.g., Shigekawa and Dower, 1988, Biotechniques 6: 742-751), orconjugation (see, e.g., Koehler and Thorne, 1987, J. Bacteriol. 169:5271-5278). The introduction of DNA into an E. coli cell may be effectedby protoplast transformation (see, e.g., Hanahan, 1983, J. Mol. Biol.166: 557-580) or electroporation (see, e.g., Dower et al., 1988, NucleicAcids Res. 16: 6127-6145). The introduction of DNA into a Streptomycescell may be effected by protoplast transformation, electroporation (see,e.g., Gong et al., 2004, Folia Microbiol. (Praha) 49: 399-405),conjugation (see, e.g., Mazodier et al., 1989, J. Bacteriol. 171:3583-3585), or transduction (see, e.g., Burke et al., 2001, Proc. Natl.Acad. Sci. USA 98: 6289-6294). The introduction of DNA into aPseudomonas cell may be effected by electroporation (see, e.g., Choi etal., 2006, J. Microbiol. Methods 64: 391-397) or conjugation (see, e.g.,Pinedo and Smets, 2005, Appl. Environ. Microbiol. 71: 51-57). Theintroduction of DNA into a Streptococcus cell may be effected by naturalcompetence (see, e.g., Perry and Kuramitsu, 1981, Infect. Immun. 32:1295-1297), protoplast transformation (see, e.g., Catt and Jollick,1991, Microbios 68: 189-207), electroporation (see, e.g., Buckley etal., 1999, Appl. Environ. Microbiol. 65: 3800-3804), or conjugation(see, e.g., Clewell, 1981, Microbiol. Rev. 45: 409-436). However, anymethod known in the art for introducing DNA into a host cell can beused.

The host cell may also be a eukaryote, such as a mammalian, insect,plant, or fungal cell.

The host cell may be a fungal cell. “Fungi” as used herein includes thephyla Ascomycota, Basidiomycota, Chytridiomycota, and Zygomycota as wellas the Oomycota and all mitosporic fungi (as defined by Hawksworth etal., In, Ainsworth and Bisby's Dictionary of The Fungi, 8th edition,1995, CAB International, University Press, Cambridge, UK).

The fungal host cell may be a yeast cell. “Yeast” as used hereinincludes ascosporogenous yeast (Endomycetales), basidiosporogenousyeast, and yeast belonging to the Fungi Imperfecti (Blastomycetes).Since the classification of yeast may change in the future, for thepurposes of this invention, yeast shall be defined as described inBiology and Activities of Yeast (Skinner, Passmore, and Davenport,editors, Soc. App. Bacteriol. Symposium Series No. 9, 1980).

The yeast host cell may be a Candida, Hansenula, Kluyveromyces, Pichia,Saccharomyces, Schizosaccharomyces, or Yarrowia cell, such as aKluyveromyces lactis, Saccharomyces carlsbergensis, Saccharomycescerevisiae, Saccharomyces diastaticus, Saccharomyces douglasii,Saccharomyces kluyveri, Saccharomyces norbensis, Saccharomycesoviformis, or Yarrowia lipolytica cell.

The fungal host cell may be a filamentous fungal cell. “Filamentousfungi” include all filamentous forms of the subdivision Eumycota andOomycota (as defined by Hawksworth et al., 1995, supra). The filamentousfungi are generally characterized by a mycelial wall composed of chitin,cellulose, glucan, chitosan, mannan, and other complex polysaccharides.Vegetative growth is by hyphal elongation and carbon catabolism isobligately aerobic. In contrast, vegetative growth by yeasts such asSaccharomyces cerevisiae is by budding of a unicellular thallus andcarbon catabolism may be fermentative.

The filamentous fungal host cell may be an Acremonium, Aspergillus,Aureobasidium, Bjerkandera, Ceriporiopsis, Chrysosporium, Coprinus,Coriolus, Cryptococcus, Filibasidium, Fusarium, Humicola, Magnaporthe,Mucor, Myceliophthora, Neocallimastix, Neurospora, Paecilomyces,Penicillium, Phanerochaete, Phlebia, Piromyces, Pleurotus,Schizophyllum, Talaromyces, Thermoascus, Thielavia, Tolypocladium,Trametes, or Trichoderma cell.

For example, the filamentous fungal host cell may be an Aspergillusawamori, Aspergillus foetidus, Aspergillus fumigatus, Aspergillusjaponicus, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae,Bjerkandera adusta, Ceriporiopsis aneirina, Ceriporiopsis caregiea,Ceriporiopsis gilvescens, Ceriporiopsis pannocinta, Ceriporiopsisrivulosa, Ceriporiopsis subrufa, Ceriporiopsis subvermispora,Chrysosporium inops, Chrysosporium keratinophilum, Chrysosporiumlucknowense, Chrysosporium merdarium, Chrysosporium pannicola,Chrysosporium queenslandicum, Chrysosporium tropicum, Chrysosporiumzonatum, Coprinus cinereus, Coriolus hirsutus, Fusarium bactridioides,Fusarium cerealis, Fusarium crookwellense, Fusarium culmorum, Fusariumgraminearum, Fusarium graminum, Fusarium heterosporum, Fusarium negundi,Fusarium oxysporum, Fusarium reticulatum, Fusarium roseum, Fusariumsambucinum, Fusarium sarcochroum, Fusarium sporotrichioides, Fusariumsulphureum, Fusarium torulosum, Fusarium trichothecioides, Fusariumvenenatum, Humicola insolens, Humicola lanuginosa, Mucor miehei,Myceliophthora thermophila, Neurospora crassa, Penicillium purpurogenum,Phanerochaete chrysosporium, Phlebia radiata, Pleurotus eryngii,Thielavia terrestris, Trametes villosa, Trametes versicolor, Trichodermaharzianum, Trichoderma koningii, Trichoderma longibrachiatum,Trichoderma reesei, or Trichoderma viride cell.

Fungal cells may be transformed by a process involving protoplastformation, transformation of the protoplasts, and regeneration of thecell wall in a manner known per se. Suitable procedures fortransformation of Aspergillus and Trichoderma host cells are describedin EP 238023, Yelton et al., 1984, Proc. Natl. Acad. Sci. USA 81:1470-1474, and Christensen et al., 1988, Bio/Technology 6: 1419-1422.Suitable methods for transforming Fusarium species are described byMalardier et al., 1989, Gene 78: 147-156, and WO 96/00787. Yeast may betransformed using the procedures described by Becker and Guarente, InAbelson, J. N. and Simon, M. I., editors, Guide to Yeast Genetics andMolecular Biology, Methods in Enzymology, Volume 194, pp. 182-187,Academic Press, Inc., New York; Ito et al., 1983, J. Bacteriol. 153:163; and Hinnen et al., 1978, Proc. Natl. Acad. Sci. USA 75: 1920.

The host cells are cultivated in a nutrient medium suitable forproduction of the xyloglucan endotransglycosylase using methods known inthe art. For example, the cells may be cultivated by shake flaskcultivation, or small-scale or large-scale fermentation (includingcontinuous, batch, fed-batch, or solid state fermentations) inlaboratory or industrial fermentors in a suitable medium and underconditions allowing the xyloglucan endotransglycosylase to be expressedand/or isolated. The cultivation takes place in a suitable nutrientmedium comprising carbon and nitrogen sources and inorganic salts, usingprocedures known in the art. Suitable media are available fromcommercial suppliers or may be prepared according to publishedcompositions (e.g., in catalogues of the American Type CultureCollection). If the xyloglucan endotransglycosylase is secreted into thenutrient medium, the polypeptide can be recovered directly from themedium. If the xyloglucan endotransglycosylase is not secreted, it canbe recovered from cell lysates.

The xyloglucan endotransglycosylase may be detected using methods knownin the art that are specific for the polypeptides. These detectionmethods include, but are not limited to, use of specific antibodies,formation of an enzyme product, or disappearance of an enzyme substrate.For example, an enzyme assay may be used to determine the activity ofthe polypeptide.

The xyloglucan endotransglycosylase may be recovered using methods knownin the art. For example, the polypeptide may be recovered from thenutrient medium by conventional procedures including, but not limitedto, collection, centrifugation, filtration, extraction, spray-drying,evaporation, or precipitation. In one aspect, a whole fermentation brothcomprising the polypeptide is recovered. In a preferred aspect,xyloglucan endotransglycosylase yield may be improved by subsequentlywashing cellular debris in buffer or in buffered detergent solution toextract biomass-associated polypeptide.

The xyloglucan endotransglycosylase may be purified by a variety ofprocedures known in the art including, but not limited to,chromatography (e.g., ion exchange, affinity, hydrophobic interaction,mixed mode, reverse phase, chromatofocusing, and size exclusion),electrophoretic procedures (e.g., preparative isoelectric focusing),differential solubility (e.g., ammonium sulfate precipitation), PAGE,membrane-filtration or extraction (see, e.g., Protein Purification,Janson and Ryden, editors, VCH Publishers, New York, 1989) to obtainsubstantially pure polypeptide. In a preferred aspect, xyloglucanendotransglycosylase may be purified by formation of a covalentacyl-enzyme intermediate with xyloglucan, followed by precipitation withmicrocrystalline cellulose or adsorption to cellulose membranes. Releaseof the polypeptide is then effected by addition of xyloglucan oligomersto resolve the covalent intermediate (Sulova and Farkas, 1999, ProteinExpression and Purification 16(2): 231-235, and Steele and Fry, 1999,Biochemical Journal 340: 207-211).

The present invention is further described by the following examplesthat should not be construed as limiting the scope of the invention.

EXAMPLES Media and Solutions

COVE agar plates were composed of 342.3 g of sucrose, 252.54 g of CsCl,59.1 g of acetamide, 520 mg of KCl, 520 mg of MgSO₄.7H₂O, 1.52 g ofKH₂PO₄, 0.04 mg of Na₂B₄O₇.10H₂O, 0.4 mg of CuSO₄.5H₂O, 1.2 mg ofFeSO₄.7H₂O, 0.7 mg of MnSO₄.2H₂O, 0.8 mg of Na₂MoO₄.2H₂O, 10 mg ofZnSO₄.7H₂O, 25 g of Noble agar, and deionized water to 1 liter.

LB medium was composed of 10 g of tryptone, 5 g of yeast extract, 5 g ofNaCl, and deionized water to 1 liter.

LB plates were composed of 10 g of tryptone, 5 g of yeast extract, 5 gof NaCl, 15 g of bacteriological agar, and deionized water to 1 liter.

Minimal medium agar plates were composed of 342.3 g of sucrose, 10 g ofglucose, 4 g of MgSO₄.7H₂O, 6 g of NaNO₃, 0.52 g of KCl, 1.52 g ofKH₂PO₄, 0.04 mg of Na₂B₄O₇.10H₂O, 0.4 mg of CuSO₄.5H₂O, 1.2 mg ofFeSO₄.7H₂O, 0.7 mg of MnSO₄.2H₂O, 0.8 mg of Na₂MoO₄.2H₂O, 10 mg ofZnSO₄.7H₂O, 500 mg of citric acid, 4 mg of d-biotin, 20 g of Noble agar,and deionized water to 1 liter.

Synthetic defined medium lacking uridine was composed of 18 mg ofadenine hemisulfate, 76 mg of alanine, 76 mg of arginine hydrochloride,76 mg of asparagine monohydrate, 76 mg of aspartic acid, 76 mg ofcysteine hydrochloride monohydrate, 76 mg of glutamic acid monosodiumsalt, 76 mg of glutamine, 76 mg of glycine, 76 mg of histidine, myo-76mg of inositol, 76 mg of isoleucine, 380 mg of leucine, 76 mg of lysinemonohydrochloride, 76 mg of methionine, 8 mg of p-aminobenzoic acidpotassium salt, 76 mg of phenylalanine, 76 mg of proline, 76 mg ofserine, 76 mg of threonine, 76 mg of tryptophan, 76 mg of tyrosinedisodium salt, 76 mg of valine, and deionized water to 1 liter.

TAE buffer was composed of 4.84 g of Tris base, 1.14 ml of glacialacetic acid, 2 ml of 0.5 M EDTA pH 8.0, and deionized water to 1 liter.

TBE buffer was composed of 10.8 g of Tris base, 5.5 g of boric acid, 4ml of 0.5 M EDTA pH 8.0, and deionized water to 1 liter.

2×YT plus ampicillin plates were composed of 16 g of tryptone, 10 g ofyeast extract, 5 g of sodium chloride, 15 g of Bacto agar, and deionizedwater to 1 liter. One ml of a 100 mg/ml solution of ampicillin was addedafter the autoclaved medium was tempered to 55° C.

YP+2% glucose medium was composed of 10 g of yeast extract, 20 g ofpeptone, 20 g of glucose, and deionized water to 1 liter.

YP+2% maltodextrin medium was composed of 10 g of yeast extract, 20 g ofpeptone, 20 g of maltodextrin, and deionized water to 1 liter.

Example 1: Preparation of Vigna angularis XyloglucanEndotransglycosylase 16

Vigna angularis xyloglucan endotransglycosylase 16 (VaXET16; SEQ ID NO:1 [native DNA sequence], SEQ ID NO: 2 [synthetic DNA sequence], and SEQID NO: 3 [deduced amino acid sequence]; also referred to as XTH1) wasrecombinantly produced in Aspergillus oryzae MT3568 according to theprotocol described below. Aspergillus oryzae MT3568 is an amdS(acetamidase) disrupted gene derivative of Aspergillus oryzae JaL355 (WO2002/40694), in which pyrG auxotrophy was restored by disrupting the A.oryzae amdS gene with the pyrG gene.

The vector pDLHD0012 was constructed to express the VaXET16 gene inmulti-copy in Aspergillus oryzae. Plasmid pDLHD0012 was generated bycombining two DNA fragments using megaprimer cloning: Fragment 1containing the VaXET16 ORF and flanking sequences with homology tovector pBM120 (US20090253171), and Fragment 2 consisting of an inversePCR amplicon of vector pBM120.

Fragment 1 was amplified using primer 613788 (sense) and primer 613983(antisense) shown below. These primers were designed to contain flankingregions of sequence homology to vector pBM120 (lower case) forligation-free cloning between the PCR fragments.

Primer 613788 (sense): (SEQ ID NO: 7)ttcctcaatcctctatatacacaactggccATGGGCTCGTCCCTGGGACPrimer 613983 (antisense): (SEQ ID NO: 8)tgtcagtcacctctagttaattaGATGTCCCTATCGCGTGTACACTCG

Fragment 1 was amplified by PCR in a reaction composed of 10 ng of aGENEART® vector pMA containing the VaXET16 synthetic gene (SEQ ID NO: 3[synthetic DNA sequence]) cloned between the Sac I and Kpn I sites, 0.5μl of PHUSION® DNA Polymerase (New England Biolabs, Inc., Ipswich,Mass., USA), 20 pmol of primer 613788, 20 pmol of primer 613983, 1 μl of10 mM dNTPs, 10 μl of 5× PHUSION® HF buffer (New England Biolabs, Inc.,Ipswich, Mass., USA), and 35.5 μl of water. The reaction was incubatedin an EPPENDORF® MASTERCYCLER® (Eppendorf AG, Hamburg, Germany)programmed for 1 cycle at 98° C. for 30 seconds; and 30 cycles each at98° C. for 10 seconds, 60° C. for 10 seconds, and 72° C. for 30 seconds.The resulting 0.9 kb PCR product (Fragment 1) was treated with 1 μl ofDpn I (Promega, Fitchburg, Wis., USA) to remove plasmid template DNA.The Dpn I was added directly to the PCR tube, mixed well, and incubatedat 37° C. for 60 minutes, and then was column-purified using a MINELUTE®PCR Purification Kit (QIAGEN Inc., Valencia, Calif., USA) according tothe manufacturer's instructions.

Fragment 2 was amplified using primers 613786 (sense) and 613787(antisense) shown below.

613786 (sense): taattaactagaggtgactgacacctggc (SEQ ID NO: 9)613787 (antisense): catggccagttgtgtatatagaggattgagg (SEQ ID NO: 10)

Fragment 2 was amplified by PCR in a reaction composed of 10 ng ofplasmid pBM120, 0.5 μl of PHUSION® DNA Polymerase, 20 pmol of primer613786, 20 pmol of primer 613787, 1 μl of 10 mM dNTPs, 10 μl of 5×PHUSION® HF buffer, and 35.5 μl of water. The reaction was incubated inan EPPENDORF® MASTERCYCLER® programmed for 1 cycle at 98° C. for 30seconds; and 30 cycles each at 98° C. for 10 seconds, 60° C. for 10seconds, and 72° C. for 4 minutes. The resulting 6.9 kb PCR product(Fragment 2) was treated with 1 μl of Dpn I to remove plasmid templateDNA. The Dpn I was added directly to the PCR tube, mixed well, andincubated at 37° C. for 60 minutes, and then column-purified using aMINELUTE® PCR Purification Kit according to the manufacturer'sinstructions.

The following procedure was used to combine the two PCR fragments usingmegaprimer cloning. Fragments 1 and 2 were combined by PCR in a reactioncomposed of 5 μl of each purified PCR product, 0.5 μl of PHUSION® DNAPolymerase, 1 μl of 10 mM dNTPs, 10 μl of 5× PHUSION® HF buffer, and28.5 μl of water. The reaction was incubated in an EPPENDORF®MASTERCYCLER® programmed for 1 cycle at 98° C. for 30 seconds; and 40cycles each at 98° C. for 10 seconds, 60° C. for 10 seconds, and 72° C.for 4 minutes. Two μl of the resulting PCR product DNA was thentransformed into E. coli ONE SHOT® TOP10 electrocompetent cells (LifeTechnologies, Grand Island, N.Y., USA) according the manufacturer'sinstructions. Fifty μl of transformed cells were spread onto LB platessupplemented with 100 μg of ampicillin per ml and incubated at 37° C.overnight. Individual transformants were picked into 3 ml of LB mediumsupplemented with 100 μg of ampicillin per ml and grown overnight at 37°C. with shaking at 250 rpm. The plasmid DNA was purified from thecolonies using a QIAPREP® Spin Miniprep Kit (QIAGEN Inc., Valencia,Calif., USA). DNA sequencing using a 3130XL Genetic Analyzer (AppliedBiosystems, Foster City, Calif., USA) was used to confirm the presenceof each of both fragments in the final plasmid pDLHD0012 (FIG. 1).

Aspergillus oryzae strain MT3568 was transformed with plasmid pDLHD0012comprising the VaXET16 gene according to the following protocol.Approximately 2-5×10⁷ spores of A. oryzae strain MT3568 were inoculatedinto 100 ml of YP+2% glucose medium in a 500 ml shake flask andincubated at 28° C. and 110 rpm overnight. Ten ml of the overnightculture were filtered in a 125 ml sterile vacuum filter, and the myceliawere washed twice with 50 ml of 0.7 M KCl-20 mM CaCl₂. The remainingliquid was removed by vacuum filtration, leaving the mat on the filter.Mycelia were resuspended in 10 ml of 0.7 M KCl-20 mM CaCl₂ andtransferred to a sterile 125 ml shake flask containing 20 mg ofGLUCANEX® 200 G (Novozymes Switzerland AG, Neumatt, Switzerland) per mland 0.2 mg of chitinase (Sigma-Aldrich, St. Louis, Mo., USA) per ml in10 ml of 0.7 M KCl-20 mM CaCl₂. The mixture was incubated at 37° C. and100 rpm for 30-90 minutes until protoplasts were generated from themycelia. The protoplast mixture was filtered through a sterile funnellined with MIRACLOTH® (Calbiochem, San Diego, Calif., USA) into asterile 50 ml plastic centrifuge tube to remove mycelial debris. Thedebris in the MIRACLOTH® was washed thoroughly with 0.7 M KCl-20 mMCaCl₂, and centrifuged at 2500 rpm (537×g) for 10 minutes at 20-23° C.The supernatant was removed and the protoplast pellet was resuspended in20 ml of 1 M sorbitol-10 mM Tris-HCl (pH 6.5)-10 mM CaCl₂. This step wasrepeated twice, and the final protoplast pellet was resuspended in 1 Msorbitol-10 mM Tris-HCl (pH 6.5)-10 mM CaCl₂ to obtain a finalprotoplast concentration of 2×10⁷/ml.

Two micrograms of pDLHD0012 were added to the bottom of a sterile 2 mlplastic centrifuge tube. Then 100 μl of protoplasts were added to thetube followed by 300 μl of 60% PEG-4000 in 10 mM Tris-HCl (pH 6.5)-10 mMCaCl₂. The tube was mixed gently by hand and incubated at 37° C. for 30minutes. Two ml of 1 M sorbitol-10 mM Tris-HCl (pH 6.5)-10 mM CaCl₂ wereadded to each transformation and the mixture was transferred onto 150 mmCOVE agar plates. Transformation plates were incubated at 34° C. untilcolonies appeared.

Twenty-one transformant colonies were picked to fresh COVE agar platesand cultivated at 34° C. for four days until the transformantssporulated. Fresh spores were transferred to 48-well deep-well platescontaining 2 ml of YP+2% maltodextrin, covered with a breathable seal,and grown for 4 days at 34° C. with no shaking. After 4 days growthsamples of the culture media were assayed for xyloglucanendotransglycosylase activity using an iodine stain assay and forxyloglucan endotransglycosylase expression by SDS-PAGE.

The iodine stain assay for xyloglucan endotransglycosylase activity wasperformed according to the following protocol. In a 96-well plate, 5 μlof culture broth were added to a mixture of 5 μl of xyloglucan(Megazyme, Bray, United Kingdom) (5 mg/ml in water), 20 μl of xyloglucanoligomers (Megazyme, Bray, United Kingdom) (5 mg/ml in water), and 10 μlof 400 mM sodium citrate pH 5.5. The reaction mix was incubated at 37°C. for thirty minutes, quenched with 200 μl of a solution containing 14%(w/v) Na₂SO₄, 0.2% KI, 100 mM HCl, and 1% iodine (I₂), incubated in thedark for 30 minutes, and then the absorbance was measured in a platereader at 620 nm. The assay demonstrated the presence of xyloglucanendotransglycosylase activity from several transformants.

SDS-PAGE was performed using a 8-16% CRITERION® Stain Free SDS-PAGE gel(Bio-Rad Laboratories, Inc., Hercules, Calif., USA), and imaging the gelwith a Stain Free Imager (Bio-Rad Laboratories, Inc., Hercules, Calif.,USA) using the following settings: 5-minute activation, automaticimaging exposure (intense bands), highlight saturated pixels=ON,color=Coomassie, and band detection, molecular weight analysis andreporting disabled. SDS-PAGE analysis indicated that severaltransformants expressed a protein of approximately 32 kDa correspondingto VaXET16.

Example 2: Construction of Plasmid pMMar27 as a Yeast Expression PlasmidVector

Plasmid pMMar27 was constructed for expression of the T. terrestrisCel6A cellobiohydrolase II in yeast. The plasmid was generated from alineage of yeast expression vectors: plasmid pMMar27 was constructedfrom plasmid pBM175b; plasmid pBM175b was constructed from plasmidpBM143b (WO 2008/008950) and plasmid pJLin201; and plasmid pJLin201 wasconstructed from pBM143b.

Plasmid pJLin201 is identical to pBM143b except an Xba I siteimmediately downstream of a Thermomyces lanuginosus lipase variant genein pBM143b was mutated to a unique Nhe I site. A QUIKCHANGE® II XLSite-Directed Mutagenesis Kit (Stratagene, La Jolla, Calif., USA) wasused to change the Xba I sequence (TCTAGA) to a Nhe I sequence (gCTAGc)in pBM143b. Primers employed to mutate the site are shown below.

Primer 999551 (sense): (SEQ ID NO: 11)5′-ACATGTCTTTGATAAgCTAGcGGGCCGCATCATGTA-3′ Primer 999552 (antisense):(SEQ ID NO: 12) 5′-TACATGATGCGGCCCgCTAGcTTATCAAAGACATGT-3′Lower case represents mutated nucleotides.

The amplification reaction was composed of 125 ng of each primer above,20 ng of pBM143b, 1× QUIKCHANGE® Reaction Buffer (Stratagene, La Jolla,Calif., USA), 3 μl of QUIKSOLUTION® (Stratagene, La Jolla, Calif., USA),1 μl of dNTP mix, and 1 μl of a 2.5 units/ml Pfu Ultra HF DNA polymerasein a final volume of 50 μl. The reaction was performed using anEPPENDORF® MASTERCYCLER® thermocycler programmed for 1 cycle at 95° C.for 1 minute; 18 cycles each at 95° C. for 50 seconds, 60° C. for 50seconds, and 68° C. for 6 minutes and 6 seconds; and 1 cycle at 68° C.for 7 minutes. After the PCR, the tube was placed on ice for 2 minutes.One microliter of Dpn I was directly added to the amplification reactionand incubated at 37° C. for 1 hour. A 2 μl volume of the Dpn I digestedreaction was used to transform E. coli XL10-GOLD® Ultracompetent Cells(Stratagene, La Jolla, Calif., USA) according to the manufacturer'sinstructions. E. coli transformants were selected on 2×YT plusampicillin plates. Plasmid DNA was isolated from several of thetransformants using a BIOROBOT® 9600. One plasmid with the desired Nhe Ichange was confirmed by restriction digestion and sequencing analysisand designated plasmid pJLin201. To eliminate possible PCR errorsintroduced by site-directed-mutagenesis, plasmid pBM175b was constructedby cloning the Nhe I site containing fragment back into plasmid pBM143b.Briefly, plasmid pJLin201 was digested with Nde I and Nu I and theresulting fragment was cloned into pBM143b previously digested with thesame enzymes using a Rapid Ligation Kit (Roche Diagnostics Corporation,Indianapolis, Ind., USA). Then, 7 μl of the Nde I/Mlu I digestedpJLin201 fragment and 1 μl of the digested pBM143b were mixed with 2 μlof 5×DNA dilution buffer (Roche Diagnostics Corporation, Indianapolis,Ind., USA), 10 μl of 2×T4 DNA ligation buffer (Roche DiagnosticsCorporation, Indianapolis, Ind., USA), and 1 μl of T4 DNA ligase (RocheDiagnostics Corporation, Indianapolis, Ind., USA) and incubated for 15minutes at room temperature. Two microliters of the ligation weretransformed into XL1-Blue Subcloning-Grade Competent Cells (Stratagene,La Jolla, Calif., USA) cells and spread onto 2×YT plus ampicillinplates. Plasmid DNA was purified from several transformants using aBIOROBOT® 9600 and analyzed by DNA sequencing using a 3130XL GeneticAnalyzer to identify a plasmid containing the desired A. nidulans pyrGinsert. One plasmid with the expected DNA sequence was designatedpBM175b.

Plasmid pMMar27 was constructed from pBM175b and an amplified gene of T.terrestris Cel6A cellobiohydrolase II with overhangs designed forinsertion into digested pBM175b. Plasmid pBM175b containing theThermomyces lanuginosus lipase variant gene under control of the CUP Ipromoter contains unique Hind III and Nhe I sites to remove the lipasegene. Plasmid pBM 175 was digested with these restriction enzymes toremove the lipase gene. After digestion, the empty vector was isolatedby 1.0% agarose gel electrophoresis using TBE buffer where anapproximately 5,215 bp fragment was excised from the gel and extractedusing a QIAQUICK® Gel Extraction Kit. The ligation reaction (20 μl) wascomposed of 1× IN-FUSION® Buffer (BD Biosciences, Palo Alto, Calif.,USA), 1×BSA (BD Biosciences, Palo Alto, Calif., USA), 1 μl of IN-FUSION®enzyme (diluted 1:10) (BD Biosciences, Palo Alto, Calif., USA), 99 ng ofpBM175b digested with Hind III and Nhe I, and 36 ng of the purified T.terrestris Cel6A cellobiohydrolase II PCR product. The reaction wasincubated at room temperature for 30 minutes. A 2 μl volume of theIN-FUSION® reaction was transformed into E. coli XL10-GOLD®Ultracompetent Cells. Transformants were selected on LB platessupplemented with 100 μg of ampicillin per ml. A colony was picked thatcontained the T. terrestris Cel6A inserted into the pBM175b vector inplace of the lipase gene, resulting in pMMar27 (FIG. 2). The plasmidchosen contained a PCR error at position 228 from the start codon, TCTinstead of TCC, but resulted in a silent change in the T. terrestrisCel6A cellobiohydrolase II.

Example 3: Construction of pEvFz1 Expression Vector

Expression vector pEvFz1 was constructed by modifying pBM120a (U.S. Pat.No. 8,263,824) to comprise the NA2/NA2-tpi promoter, A. nigeramyloglucosidase terminator sequence (AMG terminator), and Aspergillusnidulans orotidine-5′-phosphate decarboxylase gene (pyrG) as aselectable marker.

Plasmid pEvFz1 was generated by cloning the A. nidulans pyrG gene frompAILo2 (WO 2004/099228) into pBM120a. Plasmids pBM120a and pAILo2 weredigested with Nsi I overnight at 37° C. The resulting 4176 bp linearizedpBM120a vector fragment and the 1479 bp pyrG gene insert from pAILo2were each purified by 0.7% agarose gel electrophoresis using TAE buffer,excised from the gel, and extracted using a QIAQUICK® Gel ExtractionKit.

The 1479 bp pyrG gene insert was ligated to the Nsi I digested pBM120afragment using a QUICK LIGATION™ Kit (New England Biolabs, Beverly,Mass., USA). The ligation reaction was composed of 1× QUICK LIGATION™Reaction Buffer (New England Biolabs, Beverly, Mass., USA), 50 ng of NsiI digested pBM120a vector, 54 ng of the 1479 bp Nsi I digested pyrG geneinsert, and 1 μl of T4 DNA ligase in a total volume of 20 μl. Theligation mixture was incubated at 37° C. for 15 minutes followed at 50°C. for 15 minutes and then placed on ice.

One μl of the ligation mixture was transformed into ONE SHOT® TOP10chemically competent Escherichia coli cells. Transformants were selectedon 2×YT plus ampicillin plates. Plasmid DNA was purified from severaltransformants using a BIOROBOT® 9600 and analyzed by DNA sequencingusing a 3130XL Genetic Analyzer to identify a plasmid containing thedesired A. nidulans pyrG insert. One plasmid with the expected DNAsequence was designated pEvFz1 (FIG. 3).

Example 4: Construction of the Plasmid pDLHD0006 as a Yeast/E. coli/A.oryzae Shuttle Vector

Plasmid pDLHD0006 was constructed as a base vector to enable A. oryzaeexpression cassette library building using yeast recombinationalcloning. Plasmid pDLHD0006 was generated by combining three DNAfragments using yeast recombinational cloning: Fragment 1 containing theE. coli pUC origin of replication, E. coli beta-lactamase (ampR)selectable marker, URA3 yeast selectable marker, and yeast 2 micronorigin of replication from pMMar27 (Example 2); Fragment 2 containingthe 10 amyR/NA2-tpi promoter (a hybrid of the promoters from the genesencoding Aspergillus niger neutral alpha-amylase and Aspergillus oryzaetriose phosphate isomerase and including 10 repeated binding sites forthe Aspergillus oryzae amyR transcription factor), Thermomyceslanuginosus lipase open reading frame (ORF), and Aspergillus nigerglucoamylase terminator from pJaL1262 (WO 2013/178674); and Fragment 3containing the Aspergillus nidulans pyrG selection marker from pEvFz1(Example 3).

PCR pDLHD0006 PCR Contents Template Fragment 1 E. coli ori/AmpR/URA/2micron (4.1 kb) pMMar27 Fragment 2 10 amyR/NA2-tpi PR/lipase/Tamg (4.5kb) pJaL1262 Fragment 3 pyrG gene from pEvFz1 (1.7 kb) pEvFz1

Fragment 1 was amplified using primers 613017 (sense) and 613018(antisense) shown below. Primer 613017 was designed to contain aflanking region with sequence homology to Fragment 3 (lower case) andprimer 613018 was designed to contain a flanking region with sequencehomology to Fragment 2 (lower case) to enable yeast recombinationalcloning between the three PCR fragments.

Primer 613017 (sense): (SEQ ID NO: 13)ttaatcgccttgcagcacaCCGCTTCCTCGCTCACTGACTC 613018 (antisense):(SEQ ID NO: 14) acaataaccctgataaatgcGGAACAACACTCAACCCTATCTCGGTC

Fragment 1 was amplified by PCR in a reaction composed of 10 ng ofplasmid pMMar27, 0.5 μl of PHUSION® DNA Polymerase (New England Biolabs,Inc., Ipswich, Mass., USA), 20 pmol of primer 613017, 20 pmol of primer613018, 1 μl of 10 mM dNTPs, 10 μl of 5× PHUSION® HF buffer, and 35.5 μlof water. The reaction was incubated in an EPPENDORF® MASTERCYCLER®programmed for 1 cycle at 98° C. for 30 seconds; and 30 cycles each at98° C. for 10 seconds, 60° C. for 10 seconds, and 72° C. for 1.5minutes. The resulting 4.1 kb PCR product (Fragment 1) was used directlyfor yeast recombination with Fragments 2 and 3 below.

Fragment 2 was amplified using primers 613019 (sense) and 613020(antisense) shown below. Primer 613019 was designed to contain aflanking region of sequence homology to Fragment 1 (lower case) andprimer 613020 was designed to contain a flanking region of sequencehomology to Fragment 3 (lower case) to enable yeast recombinationalcloning between the three PCR fragments.

613019 (sense): (SEQ ID NO: 15)agatagggttgagtgttgttccGCATTTATCAGGGTTATTGTCTCATGAG CGG613020 (antisense): (SEQ ID NO: 16)ttctacacgaaggaaagagGAGGAGAGAGTTGAACCTGGACG

Fragment 2 was amplified by PCR in a reaction composed of 10 ng ofplasmid pJaL1262, 0.5 μl of PHUSION® DNA Polymerase, 20 pmol of primer613019, 20 pmol of primer 613020, 1 μl of 10 mM dNTPs, 10 μl of 5×PHUSION® HF buffer, and 35.5 μl of water. The reaction was incubated inan EPPENDORF® MASTERCYCLER® programmed for 1 cycle at 98° C. for 30seconds; 30 cycles each at 98° C. for 10 seconds, 60° C. for 10 seconds,and 72° C. for 2 minutes; and a 20° C. hold. The resulting 4.5 kb PCRproduct (Fragment 2) was used directly for yeast recombination withFragment 1 above and Fragment 3 below.

Fragment 3 was amplified using primers 613022 (sense) and 613021(antisense) shown below. Primer 613021 was designed to contain aflanking region of sequence homology to Fragment 2 (lower case) andprimer 613022 was designed to contain a flanking region of sequencehomology to Fragment 1 (lower case) to enable yeast recombinationalcloning between the three PCR fragments.

613022 (sense): (SEQ ID NO: 17)aggttcaactctctcctcCTCTTTCCTTCGTGTAGAAGACCAGACAG 613021 (antisense):(SEQ ID NO: 18) tcagtgagcgaggaagcggTGTGCTGCAAGGCGATTAAGTTGG

Fragment 3 was amplified by PCR in a reaction composed of 10 ng ofplasmid pEvFz1 (Example 3), 0.5 μl of PHUSION® DNA Polymerase, 20 pmolof primer 613021, 20 pmol of primer 613022, 1 μl of 10 mM dNTPs, 10 μlof 5× PHUSION® HF buffer, and 35.5 μl of water. The reaction wasincubated in an EPPENDORF® MASTERCYCLER® programmed for 1 cycle at 98°C. for 30 seconds; 30 cycles each at 98° C. for 10 seconds, 60° C. for10 seconds, and 72° C. for 2 minutes; and a 20° C. hold. The resulting1.7 kb PCR product (Fragment 3) was used directly for yeastrecombination with Fragments 1 and 2 above.

The following procedure was used to combine the three PCR fragmentsusing yeast homology-based recombinational cloning. A 20 μl aliquot ofeach of the three PCR fragments was combined with 100 μg ofsingle-stranded deoxyribonucleic acid from salmon testes (Sigma-Aldrich,St. Louis, Mo., USA), 100 μl of competent yeast cells of strain YNG318(Saccharomyces cerevisiae ATCC 208973), and 600 μl of PLATE Buffer(Sigma Aldrich, St. Louis, Mo., USA), and mixed. The reaction wasincubated at 30° C. for 30 minutes with shaking at 200 rpm. The reactionwas then continued at 42° C. for 15 minutes with no shaking. The cellswere pelleted by centrifugation at 5,000×g for 1 minute and thesupernatant was discarded. The cell pellet was suspended in 200 μl ofautoclaved water and split over two agar plates containing Syntheticdefined medium lacking uridine and incubated at 30° C. for three days.The yeast colonies were isolated from the plate using 1 ml of autoclavedwater. The cells were pelleted by centrifugation at 13,000×g for 30seconds and a 100 μl aliquot of glass beads were added to the tube. Thecell and bead mixture was suspended in 250 μl of P1 buffer (QIAGEN Inc.,Valencia, Calif., USA) and then vortexed for 1 minute to lyse the cells.The plasmid DNA was purified using a QIAPREP® Spin Miniprep Kit. A 3 μlaliquot of the plasmid DNA was then transformed into E. coli ONE SHOT®TOP10 electrocompetent cells according the manufacturer's instructions.Fifty μl of transformed cells were spread onto LB plates supplementedwith 100 μg of ampicillin per ml and incubated at 37° C. overnight.Transformants were each picked into 3 ml of LB medium supplemented with100 μg of ampicillin per ml and grown overnight at 37° C. with shakingat 250 rpm. The plasmid DNA was purified from colonies using a QIAPREP®Spin Miniprep Kit. DNA sequencing with a 3130XL Genetic Analyzer wasused to confirm the presence of each of the three fragments in a finalplasmid designated pDLHD0006 (FIG. 4).

Example 5: Preparation of Arabidopsis thaliana XyloglucanEndotransglycosylase 14

Arabidopsis thaliana xyloglucan endotransglycosylase (AtXET14; SEQ IDNO: 4 [native DNA sequence], SEQ ID NO: 5 [synthetic DNA sequence], andSEQ ID NO: 6 [deduced amino acid sequence]) was recombinantly producedin Aspergillus oryzae JaL355 (WO 2008/138835).

The vector pDLHD0039 was constructed to express the AtXET14 gene inmulti-copy in Aspergillus oryzae. Plasmid pDLHD0039 was generated bycombining two DNA fragments using restriction-free cloning: Fragment 1containing the AtXET14 ORF and flanking sequences with homology tovector pDLHD0006 (Example 4), and Fragment 2 consisting of an inversePCR amplicon of vector pDLHD0006.

Fragment 1 was amplified using primers AtXET14F (sense) and AtXET14R(antisense) shown below, which were designed to contain flanking regionsof sequence homology to vector pDLHD0006 (lower case) for ligation-freecloning between the PCR fragments.

Primer AtXET14F (sense): (SEQ ID NO: 19)ttcctcaatcctctatatacacaactggccATGGCCTGTTTCGCAACCAA ACAGAtXET14R (antisense): (SEQ ID NO: 20)agctcgctagagtcgacctaGAGTTTACATTCCTTGGGGAGACCCTG

Fragment 1 was amplified by PCR in a reaction composed of 10 ng of aGENEART® vector pMA containing the AtXET14 synthetic DNA sequence clonedbetween the Sac I and Kpn I sites, 0.5 μl of PHUSION® DNA Polymerase(New England Biolabs, Inc., Ipswich, Mass., USA), 20 pmol of primerAtXET14F, 20 pmol of primer AtXET14R, 1 μl of 10 mM dNTPs, 10 μl of 5×PHUSION® HF buffer, and 35.5 μl of water. The reaction was incubated inan EPPENDORF® MASTERCYCLER® programmed for 1 cycle at 98° C. for 30seconds; and 30 cycles each at 98° C. for 10 seconds, 60° C. for 10seconds, and 72° C. for 30 seconds. The resulting 0.9 kb PCR product(Fragment 1) was treated with 1 μl of Dpn Ito remove plasmid templateDNA. The Dpn I was added directly to the PCR tube, mixed well, andincubated at 37° C. for 60 minutes, and then column-purified using aMINELUTE® PCR Purification Kit.

Fragment 2 was amplified using primers 614604 (sense) and 613247(antisense) shown below.

614604 (sense): (SEQ ID NO: 21) taggtcgactctagcgagctcgagatc613247 (antisense): (SEQ ID NO: 22)catggccagttgtgtatatagaggattgaggaaggaagag

Fragment 2 was amplified by PCR in a reaction composed of 10 ng ofplasmid pDLHD0006, 0.5 μl of PHUSION® DNA Polymerase, 20 pmol of primer614604, 20 pmol of primer 613247, 1 μl of 10 mM dNTPs, 10 μl of 5×PHUSION® HF buffer, and 35.5 μl of water. The reaction was incubated inan EPPENDORF® MASTERCYCLER® programmed for 1 cycle at 98° C. for 30seconds; and 30 cycles each at 98° C. for 10 seconds, 60° C. for 10seconds, and 72° C. for 4 minutes. The resulting 7.3 kb PCR product(Fragment 2) was treated with 1 μl of Dpn I to remove plasmid templateDNA. Dpn I was added directly to the PCR tube, mixed well, and incubatedat 37° C. for 60 minutes, and then column-purified using a MINELUTE® PCRPurification Kit.

The two PCR fragments were combined using a GENEART® Seamless Cloningand Assembly Kit (Invitrogen, Carlsbad, Calif., USA) according tomanufacturer's instructions. Three μl of the resulting reaction productDNA were then transformed into E. coli ONE SHOT® TOP10 electrocompetentcells. Fifty μl of transformed cells were spread onto LB platessupplemented with 100 μg of ampicillin per ml and incubated at 37° C.overnight. Individual transformant colonies were picked into 3 ml of LBmedium supplemented with 100 μg of ampicillin per ml and grown overnightat 37° C. with shaking at 250 rpm. The plasmid DNA was purified fromcolonies using a QIAPREP® Spin Miniprep Kit according to themanufacturer's instructions. DNA sequencing with a 3130XL GeneticAnalyzer was used to confirm the presence of each of both fragments inthe final plasmid pDLHD0039 (FIG. 5).

Aspergillus oryzae strain JaL355 was transformed with plasmid pDLHD0039comprising the AtXET14 gene according to the following protocol.Approximately 2-5×10⁷ spores of Aspergillus oryzae JaL355 wereinoculated into 100 ml of YP+2% glucose+10 mM uridine in a 500 ml shakeflask and incubated at 28° C. and 110 rpm overnight. Ten ml of theovernight culture was filtered in a 125 ml sterile vacuum filter, andthe mycelia were washed twice with 50 ml of 0.7 M KCl-20 mM CaCl₂. Theremaining liquid was removed by vacuum filtration, leaving the mat onthe filter. Mycelia were resuspended in 10 ml of 0.7 M KCl-20 mM CaCl₂)and transferred to a sterile 125 ml shake flask containing 20 mg ofGLUCANEX® 200 G per ml and 0.2 mg of chitinase per ml in 10 ml of 0.7 MKCl-20 mM CaCl₂. The mixture was incubated at 37° C. and 100 rpm for30-90 minutes until protoplasts were generated from the mycelia. Theprotoplast mixture was filtered through a sterile funnel lined withMIRACLOTH® into a sterile 50 ml plastic centrifuge tube to removemycelial debris. The debris in the MIRACLOTH® was washed thoroughly with0.7 M KCl-20 mM CaCl₂, and centrifuged at 2500 rpm (537×g) for 10minutes at 20-23° C. The supernatant was removed and the protoplastpellet was resuspended in 20 ml of 1 M sorbitol-10 mM Tris-HCl (pH6.5)-10 mM CaCl₂. This step was repeated twice, and the final protoplastpellet was resuspended in 1 M sorbitol-10 mM Tris-HCl (pH 6.5)-10 mMCaCl₂ to obtain a final protoplast concentration of 2×10⁷/ml.

Two micrograms of pDLHD0039 were added to the bottom of a sterile 2 mlplastic centrifuge tube. Then 100 μl of protoplasts were added to thetube followed by 300 μl of 60% PEG-4000 in 10 mM Tris-HCl (pH 6.5)-10 mMCaCl₂. The tube was mixed gently by hand and incubated at 37° C. for 30minutes. Two ml of 1 M sorbitol-10 mM Tris-HCl (pH 6.5)-10 mM CaCl₂ wereadded to each transformation and the mixture was transferred onto 150 mmMinimal medium agar plates. Transformation plates were incubated at 34°C. until colonies appeared.

Thirty-five transformant colonies were picked to fresh Minimal mediumagar plates and cultivated at 34° C. for four days until the strainssporulated. Fresh spores were transferred to 48-well deep-well platescontaining 2 ml of YP+2% maltodextrin, covered with a breathable seal,and grown for 4 days at 28° C. with no shaking. After 4 days growth theculture medium was assayed for xyloglucan endotransglycosylase activityand for xyloglucan endotransglycosylase expression by SDS-PAGE.

Xyloglucan endotransglycosylase activity was measured using the iodinestain assay described in Example 1. The assay demonstrated the presenceof xyloglucan endotransglycosylase activity in several transformants.

SDS-PAGE was performed as described in Example 1. SDS-PAGE analysisindicated that several transformants expressed a protein ofapproximately 32 kDa corresponding to AtXET14.

Example 6: Generation of Fluorescein Isothiocyanate-Labeled Xyloglucan

Fluorescein isothiocyanate-labeled xyloglucan oligomers (FITC-XGOs) weregenerated by reductive amination of the reducing ends of xyloglucanoligomers (XGOs) according to the procedure described by Zhou et al.,2006, Biocatalysis and Biotransformation 24: 107-120), followed byconjugation of the amino groups of the XGOs to fluoresceinisothiocyanate isomer I (Sigma Aldrich, St. Louis, Mo., USA) in 100 mMsodium bicarbonate pH 9.0 for 24 hours at room temperature. Conjugationreaction products were concentrated to dryness in vacuo, dissolved in0.5 ml of deionized water, and purified by silica gel chromatography,eluting with a gradient from 100:0:0.04 to 70:30:1acetonitrile:water:acetic acid as mobile phase. Purity and productidentity were confirmed by evaporating the buffer, dissolving in D₂O(Sigma Aldrich, St. Louis, Mo., USA), and analysis by ¹H NMR using aVarian 400 MHz MercuryVx (Agilent, Santa Clara, Calif., USA). DriedFITC-XGOs were stored at −20° C. in the dark, and were desiccated duringthaw.

Twenty-four ml of 10 mg of tamarind seed xyloglucan (Megazyme, Bray, UK)per ml of deionized water, 217 μl of 7.9 mg of FITC-XGOs per ml ofdeionized water, 1.2 ml of 400 mM sodium citrate pH 5.5, and 600 μl of1.4 mg of VaXET16 per ml of 20 mM sodium citrate pH 5.5 were mixedthoroughly and incubated overnight at room temperature. Followingovernight incubation, FITC-XG was precipitated by addition of ice coldethanol to a final volume of 110 ml, mixed thoroughly, and incubated at4° C. overnight. The precipitated FITC-XG was washed with water and thentransferred to Erlenmeyer bulbs. Residual water and ethanol were removedby evaporation using an EZ-2 Elite evaporator (SP Scientific/Genevac,Stone Ridge, N.Y., USA) for 4 hours. Dried samples were dissolved inwater, and the volume was adjusted to 48 ml with deionized water togenerate a final FITC-XG concentration of 5 mg per ml with an expectedaverage molecular weight of 100 kDa.

Example 7: Fluorescence Polarization Assay for XyloglucanEndotransglycosylation Activity

Xyloglucan endotransglycosylation activity was assessed using thefollowing assay. Reactions of 200 μl containing 1 mg of tamarind seedxyloglucan per ml, 0.01 mg/ml FITC-XGOs prepared as described in Example6, and 10 μl of appropriately diluted XET were incubated for 10 minutesat 25° C. in 20 mM sodium citrate pH 5.5 in opaque 96-well microtiterplates. Fluorescence polarization was monitored continuously over thistime period, using a SPECTRAMAX® M5 microplate reader (MolecularDevices, Sunnyvale, Calif., USA) in top-read orientation with anexcitation wavelength of 490 nm, an emission wavelength of 520 nm, a 495cutoff filter in the excitation path, high precision (100 reads), andmedium photomultiplier tube sensitivity. XET-dependent incorporation offluorescent XGOs into non-fluorescent xyloglucan (XG) results inincreasing fluorescence polarization over time. The slope of the linearregions of the polarization time progress curves was used to determinethe activity.

Example 8: Purification of Vigna angularis XyloglucanEndotransglycosylase 16

One liter solutions of crude fermentation broth of Vigna angularis werefiltered using a 0.22 μm STERICUP® filter (Millipore, Bedford, Mass.,USA) and the filtrates were stored at 4° C. Cell debris was resuspendedin 1 liter of 0.25% TRITON® X-100(4-(1,1,3,3-tetramethylbutyl)phenyl-polyethylene glycol; Sigma Aldrich,St. Louis, Mo., USA)-20 mM sodium citrate pH 5.5, incubated at least 30minutes at room temperature, and then filtered using a 0.22 μm STERICUP®filter. The filtrates containing Vigna angularis xyloglucanendotransglycosylase 16 (VaXET16) were pooled and concentrated to avolume between 500 and 1500 ml using a VIVAFLOW® 200 tangential flowconcentrator (Millipore, Bedford, Mass., USA) equipped with a 10 kDamolecular weight cutoff membrane.

The concentrated filtrates were loaded onto a 150 ml Q SEPHAROSE® BigBeads column (GE Healthcare Lifesciences, Piscataway, N.J., USA),pre-equilibrated with 20 mM sodium citrate pH 5.5, and elutedisocratically with the same buffer. The eluent was loaded onto a 75 mlPhenyl SEPHAROSE® HP column (GE Healthcare Lifesciences, Piscataway,N.J., USA) pre-equilibrated in 20% ethylene glycol-20 mM sodium citratepH 5.5. VaXET16 was eluted using a linear gradient from 20% to 50% of70% ethylene glycol in 20 mM sodium citrate pH 5.5 over 4 columnvolumes.

Purified VaXET16 was quantified using a BCA assay (Pierce, Rockford,Ill., USA) in a 96-well plate format with bovine serum albumin (Pierce,Rockford, Ill., USA) as a protein standard at concentrations between 0and 2 mg/ml and was determined to be 1.40 mg/ml. VaXET16 homogeneity wasconfirmed by the presence of a single band at approximately 32 kDa usinga 8-16% gradient CRITERION® Stain Free SDS-PAGE gel, and imaging the gelwith a Stain Free Imager using the following settings: 5-minuteactivation, automatic imaging exposure (intense bands), highlightsaturated pixels=ON, color=Coomassie, and band detection, molecularweight analysis and reporting disabled.

The activity of the purified VaXET16 was determined by measuring therate of incorporation of fluorescein isothiocyanate-labeled xyloglucanoligomers into tamarind seed xyloglucan (Megazyme, Bray, UK) byfluorescence polarization (as described in Example 7). The apparentactivity was 18.5±1.2 P s⁻¹mg⁻¹.

The purified VaXET16 preparation was tested for background enzymeactivities including xylanase, amylase, cellulase, beta-glucosidase,protease, amyloglucosidase, and lipase using standard assays as shownbelow.

Xylanase activity was assayed using wheat arabinoxylan as substrate atpH 6.0 and 50° C. Xylan hydrolysis was assessed colorimetrically at 405nm by addition of alkaline solution containing PHBAH. One FXU(S) isdefined as the endoxylanase activity using Shearzyme® (Novozymes A/S) asa standard.

Amylase activity was assayed using starch as substrate at pH 2.5 and 37°C. Starch hydrolysis was assessed by measuring the residual starchcolorimetrically at 600 nm by addition of iodine solution. One FAU(A) isdefined as the acid alpha-amylase activity using acid fungalalpha-amylase (available from Novozymes A/S) as a standard.

Amylase activity was assayed using(4,6-ethylidene(G7)-p-nitrophenyl(G1)-α,D-maltoheptaoside(4,6-ethylidene-G7-pNP) as substrate at pH 7 and 37° C. Hydrolysis ofthe substrate produces p-nitrophenol, and was assessed colorimetricallyat 405 nm. One FAU(F) is defined as fungal alpha-amylase units usingFungamyl® (Novozymes A/S) as a standard.

Cellulase activity was assayed using carboxymethylcellulose (CMC) assubstrate at pH 5.0 and 50° C. CMC hydrolysis was assessedcolorimetrically at 405 nm by addition of an alkaline solutioncontaining para-hydroxybenzoic add hydrazide (PHBAH). One CNU(B) isdefined as the cellulase activity using NS22084 enzyme (Novozymes A/S)as a standard.

Beta-glucosidase activity was assayed using cellobiose as substrate atpH 5.0 and 50° C. Production of glucose from cellobiose was assessedusing a coupled enzyme assay with hexokinase and glucose-6-phosphatedehydrogenase converting glucose to 6-phosphoglucanate followingreduction of NAD to NADH at 340 nm. One CBU(B) is defined as the amountof enzyme which releases 2 pmole of glucose per minute using cellobiaseas a standard.

The protease assay was performed using an EnzChek® Protease Assay Kit(green fluorescence) (Life Technologies, Inc., Grand Island, N.Y., USA)with casein as substrate at pH 6 or 9 and ambient temperature. One KMTUis defined as a kilo microbial trypsin unit related to the amount ofenzyme that produces 1 pmole of p-nitroaniline per minute.

Amyloglucosidase activity was assayed using maltose as substrate at pH4.3 and 37° C. Conversion of maltose to glucose was assessed using acoupled enzyme assay with hexokinase and glucose-6-phosphatedehydrogenase converting glucose to 6-phosphoglucanate followingreduction of NAD to NADH at 340 nm. One AGU is defined asamyloglucosidase units using AMG® (Novozymes A/S) as a standard.

The 4-methylumbelliferyl beta-D-lactoside (MUL) assay was performed atpH 7 and ambient temperature and measured fluorometrically at 360 nmexcitation and 465 nm emission.

Lipase activity was assayed using 4-nitropenyl butyrate (pNP-butyrate)as substrate at pH 7.5 and ambient temperature. pNP-butyrate hydrolysiswas assessed colorimetrically following p-nitrophenol release at 405 nm.One LU is defined as the amount of enzyme which releases 1 μmole oftitratable butyric acid using LIPOLASE® (Novozymes A/S) as a standard.

Additional Assay Activity Activity Assay Substrate Dilution UnitsUnits/ml Xylanase Wheat 4-fold FXU(S) ND FXU(S) arabinoxylan AmylaseFAU(A) Starch 4-fold FAU(A) ND Amylase FAU(F) Ethylidene-G7- 4-foldFAU(F) ND pNp Cellulase CMC 4-fold CNU(B) ND CNU(B) Beta-gluco-Cellobiose 4-fold CBU(B) ND sidase CBU(B) Protease, pH 6 Casein noneKMTU 740 (EnzCheck) Protease, pH 9 Casein none KMTU 332 (EnzCheck)Amylogluco- Maltose 4-fold AGU ND sidase AGU MUL MUL none Unitless NDLipase pNP-Butyrate none LU 0.02

Example 9: Purification of Arabidopsis thaliana XyloglucanEndotransglycosylase 14

The purification and quantification of the Arabidopsis thalianaxyloglucan endotransglycosylase 14 (AtXET14) was performed as describedfor VaXET16 in Example 8, except that elution from the Phenyl SEPHAROSE®HP column was performed using a linear gradient from 40% to 90% of 70%ethylene glycol in 20 mM sodium citrate pH 5.5 over 4 column volumes.

AtXET14 homogeneity was confirmed by the presence of a single band atapproximately 32 kDa using a 8-16% CRITERION® Stain Free SDS-PAGE gel,and imaging the gel with a Stain Free Imager using the followingsettings: 5-minute activation, automatic imaging exposure (intensebands), highlight saturated pixels=ON, color=Coomassie, and banddetection, molecular weight analysis and reporting disabled.

Purified AtXET14 was quantified using a BCA assay in a 96-well plateformat with bovine serum albumin as a protein standard at concentrationsbetween 0 and 2 mg/ml and was determined to be 1.49 mg/ml.

The activity of the purified AtXET14 was determined as described inExample 7. The apparent activity was 34.7±0.9 P s⁻¹ mg⁻¹.

The purified AtXET14 preparation was tested for background activitiesincluding xylanase, amylase, cellulase, beta-glucosidase, protease,amyloglucosidase, and lipase using standard assays as shown below. Thestandard assays are described in Example 8.

Additional Assay Activity Activity Assay Substrate Dilution UnitsUnits/ml Xylanase Wheat 4-fold FXU(S) ND FXU(S) arabinoxylan AmylaseFAU(A) Starch 4-fold FAU(A) ND Amylase FAU(F) Ethylidene-G7- 4-foldFAU(F) ND pNp Cellulase CMC 4-fold CNU(B) ND CNU(B) Beta-gluco-Cellobiose 4-fold CBU(B) ND sidase CBU(B) Protease, pH 6 Casein noneKMTU 82 (EnzCheck) Protease, pH 9 Casein none KMTU 53 (EnzCheck)Amylogluco- Maltose 4-fold AGU ND sidase AGU MUL MUL none Unitless NDLipase pNP-Butyrate none LU 0.24

Example 10: Prevention of Fruit Dehydration by Xyloglucan and Xyloglucanwith Arabidopsis thaliana Xyloglucan Endotransglycosylase 14

Carnation stems and banana stems (Chiquita organic) were cut into thinsections and Granny Smith apples (Yakima Fresh) were cut into smallslices using a razor blade. A 0.5 cm length at the end of each stem wasdiscarded, and the remaining sections were either dipped in a solutionof 5 mg of tamarind seed xyloglucan (Megazyme, Bray, UK) per ml of 20 mMsodium citrate pH 5.5 and the excess xyloglucan removed by touching thestem to the side of the container, or were not dipped. Several flowerstems were used, and sections from each stem were divided into bothdipped and not-dipped groups to control for stem to stem variation. Eachsection was then incubated on its side, cut ends not touching the bottomof the well, in either a CoStar 3513 12-well or CoStar 3524 24-well,flat bottomed, covered cell culture plate (Corning, Tewksbury, Mass.,USA). The samples were incubated at room temperature for 5 days.Photographs were taken at 1, 2, and 5 days to illustrate the extent ofdesiccation/oxidation.

FIG. 6 shows carnation stems dipped in xyloglucan in the upper row, ornot dipped in the lower row, following 1 day of incubation at roomtemperature. The xyloglucan dipped carnation stems appeared smooth andhydrated, whereas the carnation stems that were not dipped appeareddessicated, with white, scaly, dry patches. After 1 day of incubation,carnation stems that had been dipped in xyloglucan appearedsubstantially more hydrated than those that had not been dipped. By 2days of incubation, both dipped and not dipped stem slices appearedsimilarly dry, thus the xyloglucan reduced the rate of carnation stemdehydration. No qualitative differences between banana stems dipped andnot dipped were observed, though these were cut more thickly than thecarnations.

FIG. 7A shows apple slices dipped in xyloglucan in the upper row, or notdipped in the lower row, after 2 days of incubation; FIG. 7B shows thesame slices after 5 days of incubation. A clear reduction in the extentto which apple flesh was browned or oxidized was observed in the slicesdipped in xyloglucan after 2 days of incubation. By 5 days ofincubation, the apple slices not dipped showed indications of mold andsubstantial oxidation, whereas xyloglucan dipped slices showed onlymodest oxidation. By comparison, the extent of oxidation was similarbetween the dipped apple slices at 5 days of incubation and the slicesthat were not dipped at 2 days of incubation. These images indicate thatxyloglucan substantially slowed or prevented the oxidative damage of cutfruit, and prevented the growth of microorganisms that contribute toproduce rot.

Example 11: Preservation of Apple Freshness by Xyloglucan and Xyloglucanwith Vigna angularis Xyloglucan Endotransglycosylase 16

To generate uniformly-sized apple slices without skins, Granny Smithapples were pierced with a size 7 rubber stopper boring tool. The appleinside was removed from the boring tool and then sectioned into 1-2 mmthick discs with a razor blade. Six to eight discs were then dipped into5 ml of 40 mM sodium citrate pH 5.5 containing either 4.5 mg ofxyloglucan per ml with 35 μg of VaXET16 per ml, 4.5 mg of xyloglucan perml without VaXET16, or 5 ml of deionized water. The excess solution wasremoved from each apple slice by touching the slice to the side of thecontainer, and the apple discs were transferred to CoStar #3513 12-well,flat bottomed, covered cell culture plates using tweezers. The sampleplates were covered and incubated under ambient conditions. Images ofthe slices were taken after 3, 4, and 7 days by inverting the plates andphotographing the slices through the bottom of the plate.

FIG. 8A shows the apple slices after 3 days of incubation.

FIG. 8B shows the apple slices after 4 days of incubation.

FIG. 8C shows the apple slices after 7 days of incubation.

By 3-4 days of incubation under ambient conditions after the appleslices were prepared, substantial differences were apparent. Appleslices, initially white, had browned and oxidized surfaces in all casesand dark brown spots were evident on all slices to various degrees. Theapple slices dipped in a solution of xyloglucan and VaXET16 showed thesmallest extent of oxidation or browning of the apple flesh, whereas thesodium citrate buffer dipped and not dipped apple slices were mostoxidized. The apples not dipped appeared smaller in diameter than theother samples, indicating that they were more dehydrated than the dippedsamples.

By 7 days of incubation, all slices appeared smaller in diameterindicating they had dried out. The dark brown spots covered most of thesurface area of the xyloglucan dipped apple slices, the buffer dippedslices appeared to be moldy, and the slices that were not dippedappeared less oxidized than at previous time points but highlydesiccated. The xyloglucan and VaXET16 dipped apple slices, bycomparison, were oxidized, but less so than the xyloglucan or bufferdipped samples.

These images indicate that over 3 to 4 days, xyloglucan or particularlyxyloglucan and VaXET16 reduced the rate of apple oxidation. Over longertime periods, xyloglucan or xyloglucan and VaXET16 delayed spoilage ofcut produce.

Example 12: Quantitative Analysis of Apple Slice Images to Determine theExtent to which Xyloglucan and Vigna angularis XyloglucanEndotransglycosylase 16 Prevent Apple Oxidation

Photographs of the apple slices shown in FIGS. 9A, 9B, and 9C werequantitatively analyzed using MATLAB® (The Mathworks, Natick, Mass.,USA) to differentiate between the extent of oxidation in the appleslices dipped in xyloglucan, xyloglucan with VaXET16, 40 mM sodiumcitrate pH 5.5, or not dipped. Browning or oxidation of the applesamples was apparent as both a time-dependent browning of the whiteapple slice overall, and as an increase in the number or size of muchdarker brown spots. Additionally, in several of the images, particularlyday 7 for sodium citrate dipped apples, additional blackening wasobserved. The extent of browning and its prevention by xyloglucan andVaXET16 were quantified according to the following protocol. Individualcolor channels of the image files were examined, and the blue channelwas determined to have the greatest differences in intensity betweenboth the dark brown spots and the lighter regions of the slices and thesamples from the plates. Subsequent analysis was performed using theblue channel only. Photographs were reduced to blue channel intensityvalues and were inverted by subtraction of the maximum pixel intensityover the image. Regions of interest containing each apple sample wereselected, and a threshold filter was applied to remove non-sample pixelsfrom the region of interest. Threshold filters were held constant acrossall samples in a single plate image, but were varied between images. Foreach filtered region of interest, histograms of thethreshold-subtracted, non-zero pixel intensities were generated andmaximum likelihood estimations of best-fit parameters to single anddouble normal distributions were determined. All non-zero pixelintensities for the 8 apple slices treated in the same way werecombined, to generate a global intensity histogram for each treatment.These histograms were similarly fit by maximum likelihood estimation tosingle and double normal distributions. The extent of browning was thendetermined in 2 ways. First, as the samples show a tendency to darken oryellow overall, the mean of the single normal distribution was used todetermine the average darkness in color of all the sample pixels abovethe threshold. Second, to quantify the extent to which dark spots coverthe surface of the apple slices, the relative areas of dark spots tototal surface areas were determined. The probability distribution wasintegrated over all intensities >1×σ above the mean of the distributionto determine the relative surface area of the darkest spots.

FIG. 9A shows the pixel intensity histogram of the apple slices notdipped after 4 days of incubation. The histogram is fit to a doublenormal distribution.

FIG. 9B shows the pixel intensity histogram of the apple slices dippedin 40 mM sodium citrate pH 5.5 after 4 days of incubation. The histogramis fit to a double normal distribution.

FIG. 9C shows the pixel intensity histogram of the apple slices dippedin xyloglucan after 4 days of incubation. The histogram is fit to adouble normal distribution.

FIG. 9D shows the pixel intensity histogram of the apple slices dippedin xyloglucan and VaXET16 after 4 days of incubation. The histogram isfit to a double normal distribution.

Comparing the histograms in FIG. 9, the samples treated with xyloglucanand VaXET16 had a distribution of intensities that peaked at the lowestintensity, hence were the least yellowed. It was also evident that asubstantial fraction of pixels had intensities greater than 100 units inall samples except for those dipped in xyloglucan and VaXET16; for thexyloglucan-dipped and not-dipped apples these appeared as an additionaloverlapped distribution. By 7 days, this additional distribution wasmore evident, and was attributed to the onset of very dark spots, whichwere most apparent in the buffer and xyloglucan-dipped samples (FIG.9C). This distribution was not present in the xyloglucan andVaXET16-dipped apples, consistent with the lack of dark brown spots onthese apple slices.

FIG. 9E shows a plot of the mean of the single Gaussian distribution asa function of time for the variously treated apple slices. Apple slicesnot dipped are shown as circles, apple slices dipped in 40 mM sodiumcitrate pH 5.5 are shown as squares, apple slices dipped in xyloglucanare shown as diamonds, and apple slices dipped in xyloglucan and VaXET16are shown as triangles. With the exception of the slices not dipped, themeans increased over time, indicating that the slices became darker incolor with time. At the 3 day time point, the means of the sodiumcitrate dipped and not dipped slices were much higher than were thexyloglucan and xyloglucan with VaXET16 dipped slices. The meanintensities were 68.91±0.0666 for not dipped slices, 69.48±0.0526 forbuffer dipped slices, 47.57±0.0436 for the xyloglucan dipped slices, and50.10±0.0451 for xyloglucan and VaXET16 dipped slices. Thus dipping ineither xyloglucan or xyloglucan and VaXET16 reduced the extent ofbrowning by 45% and 38%, respectively.

From the fits of the double normal distributions, apple slices treatedin all manners had an intensity distribution mean between 50-65 unitsand a standard deviation of 20-25. Thus pixel intensities >90 wereconsidered to be outliers or to belong to the high-intensitydistribution; they were attributed to the dark brown spots. The totalnumber of pixels exceeding this intensity relative to the total numberof pixels exceeding the threshold filter gave the relative proportion ofsurface area covered by dark brown oxidation spots. This was determinedby integration of the intensity distribution over those values ofintensity, relative to the integral over all intensities and the valuesare provided in Table 1.

From the quantification of the dark brown spots, it is clear thatxyloglucan and particularly xyloglucan with VaXET16 prevented theformation of dark brown oxidation spots. The relative surface areacovered with these spots was approximately 14-fold lower at 3 days,17-fold lower at 4 days, and 7-fold lower at 7 days between thexyloglucan+VaXET16 dipped apple slices and the average of thebuffer-dipped and not-dipped apple slices.

TABLE 1 Relative surface areas of dark brown oxidation spots Day 3 Day 4Day 7 (%) (%) (%) Not dipped 12.63 20.29 1.51 Citrate dipped 14.17 17.3628.61 XG dipped 6.42 2.92 15.66 XG + XET dipped 1.77 2.03 1.87

Example 13: Preservation of Potato and Avocado Slices by Xyloglucan andXyloglucan with Arabidopsis thaliana Xyloglucan Endotransglycosylase 14

Preservation of various fruits and vegetables was assessed as describedin Example 11 with the following exceptions. Arabidopsis thalianaendotransglycosylase 14 (AtXET14) was purified as described in Example9. Eight replicate samples were dipped into either 20 mM sodium citratepH 5.5, 1 mg of tamarind seed xyloglucan per ml in 20 mM sodium citratepH 5.5, or 1 mg of tamarind seed xyloglucan per ml with 1 μM AtXET14 in20 mM sodium citrate pH 5.5. A size 7 rubber stopper boring tool wasused to generate uniformly sized cylinders of the fruit or vegetableexamined; potatoes were pierced through the entire thickness of thepotato, avocados had their pits removed and were pierced from the pithole to the skin. Cylinders were removed from the boring tool andpotatoes were sectioned into approximately 1 mm thick discs, excludingthe outer 1 cm of each cylinder. Avocado slices from equivalent depthswithin the fruit were generated in the following manner. Three cylindersof equivalent length were positioned together aligned by distance fromthe pit, and sectioned concurrently into 2 mm thick discs. The resulting3 slices from each depth were dipped differently and compared with eachother to account for potential differences in oxidation, browning, ordesiccation that may arise from differences in the fruit. At 0, 2.5, 5,21 and 70 hours of incubation under ambient conditions, culture plateswere photographed to document the degree to which the avocado and potatoslices had oxidized.

FIG. 10A shows variously treated potato slices after 0, 2.5, 5 and 21hours of incubation. From the photographs, it is evident that at thebeginning of the incubation (time=0 hour), all slices are white. Withinthe first few hours, potato slices begin to turn brown or oxidize andbecome increasingly darker in color with time. The slices that weredipped in xyloglucan and AtXET14 show the latest onset of browning, andthe smallest extent of browning at the longer incubation times.

FIG. 10B shows variously treated avocado slices after 0 and 70 hours ofincubation. The avocado fruit transitions in color from green throughyellow at increasing depths from the skin to the pit. Consequently,slices are compared at equivalent depths and hence equivalent initialcolors as is evident in the images taken at the beginning of theincubation (time=0). After 70 hours of incubation, the avocado sliceshave all browned and darkened; those dipped in xyloglucan andparticularly xyloglucan with AtXET14 are less browned.

Example 14: Fluorescein Isothiocyanate-Labeled Xyloglucan ConfirmsAssociation of Xyloglucan with Cut Fruit and Vegetables

To confirm that xyloglucan was associating with cut fruit, 20 μl ofFITC-XG or 40 mM sodium citrate pH 5.5 were applied to carnation stem,banana stem, squash stem, or apple slices, prepared as described inExample 6. Samples were incubated at room temperature for 30 minutes andimaged using a hand-held UV lamp. By visual inspection, fluorescencecould only be delineated for the squash stem that had FITC-XG applied.The other samples were too reflective, too autofluorescent, or hadinsufficiently concentrated fluorescent xyloglucan to differentiateFITC-XG fluorescence from background.

Samples were each covered with 500 μl of 40 mM sodium citrate pH 5.5 and10 μl of 1.5 mg of AtXET14 per ml of 40 mM sodium citrate pH 5.5 wereadded to each, generating 30 μg per ml final AtXET14 concentration.Samples were incubated overnight at room temperature with shaking.Following overnight incubation, samples were washed 3 times in 2 ml of150 mM sodium chloride in 20 mM phosphate pH 7.2, over a period of 8hours. Samples were then incubated overnight in a minimum volume of 150mM sodium chloride in 20 mM phosphate pH 7.0.

Thin sections of each sample were cut using a razor blade and laid ontoa FisherFinest Premium 3″×1″×1 mm microscope slide (Fisher Scientific,Inc., Pittsburgh, Pa., USA). Approximately 20 μl of deionized water wereapplied to the slide around the sample and the sample was covered with aFisherbrand 22×22-1.5 microscope coverslip (Fisher Scientific, Inc.,Pittsburgh, Pa., USA) before sealing the coverslip to the slide usingnail polish.

Laser scanning confocal microscopy was performed using an Olympus FV1000laser scanning confocal microscope (Olympus, Center Valley, Pa., USA).Data were acquired utilizing the 488 nm line of an argon ion laserexcitation source with either a 10× air gap or a 40× oil immersionobjective lens. All images were obtained using the same excitationintensity and PMT voltage; hence relative fluorescence intensities werecomparable between images.

FIG. 11 shows a series of laser scanning confocal microscope images thatcompare a fruit, flower, or vegetable incubated with AtXET14 in 150 mMsodium chloride in 20 mM phosphate pH 7.2 to incubation with AtXET14 andFITC-XG in 150 mM sodium chloride in 20 mM phosphate pH 7.2. In eachcase, FITC-XG and AtXET14 incubated samples showed much higherfluorescence intensity than did the samples incubated with only AtXET14,indicating substantial FITC-XG binding.

FIG. 11A shows a confocal image of a section of an apple slice incubatedwith AtXET14.

FIG. 11B shows a confocal image of a section of an apple slice incubatedwith AtXET14 with FITC-XG.

FIG. 11C shows a confocal image of a section of a carnation stemincubated with AtXET14.

FIG. 11D shows a confocal image of a section of a carnation stemincubated with AtXET14 with FITC-XG.

FIG. 11E shows a confocal image of a section of a banana stem incubatedwith AtXET14.

FIG. 11F shows a confocal image of a section of a banana stem incubatedwith AtXET14 with FITC-XG.

FIG. 11G shows a confocal image of a section of a squash stem incubatedwith AtXET14.

FIG. 11H shows a confocal image of a section of a squash stem incubatedwith AtXET14 and FITC-XG.

In each case the confocal microscopy image indicates that thefluorescein isothiocyanate-labeled xyloglucan associated with the cutfruit, flower or vegetable in the presence of AtXET14.

The present invention is further described by the following numberedparagraphs:

[1] A method for modifying an agricultural crop comprising treating theagricultural crop with a composition selected from the group consistingof (a) a composition comprising a xyloglucan endotransglycosylase, apolymeric xyloglucan, and a functionalized xyloglucan oligomercomprising a chemical group; (b) a composition comprising a xyloglucanendotransglycosylase, a polymeric xyloglucan functionalized with achemical group, and a functionalized xyloglucan oligomer comprising achemical group; (c) a composition comprising a xyloglucanendotransglycosylase, a polymeric xyloglucan functionalized with achemical group, and a xyloglucan oligomer; (d) a composition comprisinga xyloglucan endotransglycosylase, a polymeric xyloglucan, and axyloglucan oligomer; (e) a composition comprising a xyloglucanendotransglycosylase and a polymeric xyloglucan functionalized with achemical group; (f) a composition comprising a xyloglucanendotransglycosylase and a polymeric xyloglucan; (g) a compositioncomprising a xyloglucan endotransglycosylase and a functionalizedxyloglucan oligomer comprising a chemical group; (h) a compositioncomprising a xyloglucan endotransglycosylase and a xyloglucan oligomer,and (i) a composition of (a), (b), (c), (d), (e), (f), (g), or (h)without a xyloglucan endotransglycosylase, in a medium under conditionsleading to a modified agricultural crop possessing an improved propertycompared to the unmodified agricultural crop.

[2] The method of paragraph 1, wherein the agricultural crop isharvested.

[3] The method of paragraph 1, wherein the agricultural crop is notharvested.

[4] The method of any of paragraphs 1-3, wherein the agricultural cropis a fruit.

[5] The method of any of paragraphs 1-3, wherein the agricultural cropis a vegetable.

[6] The method of any of paragraphs 1-3, wherein the agricultural cropis a flower.

[7] The method of any of paragraphs 1-3, wherein the agricultural cropis a spice.

[8] The method of any of paragraphs 1-7, wherein the improved propertyis one or more improvements selected from the group consisting ofreducing or preventing oxidative browning, dehydration, desiccation,bacterial, fungal, microbial, animal, or insect pest infestation,senescence, early ripening, and softening; prevention of bruising,resistance to crushing, prevention or enhancement of clustering,aggregation, or association; resistance to adverse environmentalfactors; appearance, and taste, and resistance to sun or UV damage.

[9] The method of any of paragraphs 1-8, wherein the average molecularweight of the polymeric xyloglucan or the polymeric xyloglucanfunctionalized with a chemical group ranges from 2 kDa to about 500 kDa.

[10] The method of any of paragraphs 1-9, wherein the average molecularweight of the xyloglucan oligomer or the functionalized xyloglucanoligomer comprising a chemical group ranges from 0.5 kDa to about 500kDa

[11] The method of any of paragraphs 1-10, wherein the xyloglucanendotransglycosylase is present at a concentration of about 0.1 nM toabout 1 mM.

[12] The method of any of paragraphs 1-11, wherein the polymericxyloglucan or the polymeric xyloglucan functionalized with a chemicalgroup is present at about 1 ng per g of the agricultural crop to about 1g per g of the agricultural crop.

[13] The method of any of paragraphs 1-12, wherein the xyloglucanoligomer or the functionalized xyloglucan oligomer is present with thepolymeric xyloglucan at about 50:1 molar ratio to about 0.5:1 xyloglucanoligomer or functionalized xyloglucan oligomer to polymeric xyloglucan.

[14] The method of any of paragraphs 1-13, wherein the concentration ofpolymeric xyloglucan, the polymeric xyloglucan functionalized with achemical group, the xyloglucan oligomer, or the functionalizedxyloglucan oligomer comprising a chemical group incorporated into thematerial is about 0.01 g to about 500 mg per g of the agricultural crop.

[15] The method of any of paragraphs 1-14, wherein the xyloglucanoligomer or the functionalized xyloglucan oligomer is present withoutpolymeric xyloglucan or polymeric xyloglucan functionalized with achemical group at about 1 ng per g of the material to about 1 g per g ofthe agricultural crop.

[16] The method of any of paragraphs 1-15, wherein the chemical group isa compound of interest or a reactive group such as an aldehyde group, anamino group, an aromatic group, a carboxyl group, a halogen group, ahydroxyl group, a ketone group, a nitrile group, a nitro group, asulfhydryl group, or a sulfonate group.

[17] The method of any of paragraphs 1-16, wherein the xyloglucanendotransglycosylase is obtainable from a plant.

[18] The method of paragraph 17, wherein the plant is selected from thegroup consisting of a dicotyledon and a monocotyledon.

[19] The method of paragraph 18, wherein the dicotyledon is selectedfrom the group consisting of azuki beans, cauliflowers, cotton, poplaror hybrid aspen, potatoes, rapes, soy beans, sunflowers, thalecress,tobacco, and tomatoes.

[20] The method of paragraph 18, wherein the monocotyledon is selectedfrom the group consisting of wheat, rice, corn and sugar cane.

[21] The method of any of paragraphs 1-20, wherein the xyloglucanendotransglycosylase is produced by aerobic cultivation of a transformedhost organism containing the appropriate genetic information from aplant.

[22] A modified agricultural crop obtained by the method of any ofparagraphs 1-21.

[23] A modified agricultural crop comprising comprising (a) a polymericxyloglucan, and a functionalized xyloglucan oligomer comprising achemical group; (b) a polymeric xyloglucan functionalized with achemical group and a functionalized xyloglucan oligomer comprising achemical group; (c) a polymeric xyloglucan functionalized with achemical group, and a xyloglucan oligomer; (d) a polymeric xyloglucan,and a xyloglucan oligomer; (e) a polymeric xyloglucan functionalizedwith a chemical group; (f) a polymeric xyloglucan; (g) a functionalizedxyloglucan oligomer comprising a chemical group; or (h) a xyloglucanoligomer, wherein the modified agricultural crop possesses an improvedproperty compared to the unmodified agricultural crop.

[24] A composition selected from the group consisting of (a) acomposition comprising a xyloglucan endotransglycosylase, a polymericxyloglucan, and a functionalized xyloglucan oligomer comprising achemical group; (b) a composition comprising a xyloglucanendotransglycosylase, a polymeric xyloglucan functionalized with achemical group, and a functionalized xyloglucan oligomer comprising achemical group; (c) a composition comprising a xyloglucanendotransglycosylase, a polymeric xyloglucan functionalized with achemical group, and a xyloglucan oligomer; (d) a composition comprisinga xyloglucan endotransglycosylase, a polymeric xyloglucan, and axyloglucan oligomer; (e) a composition comprising a xyloglucanendotransglycosylase and a polymeric xyloglucan functionalized with achemical group; (f) a composition comprising a xyloglucanendotransglycosylase and a polymeric xyloglucan; (g) a compositioncomprising a xyloglucan endotransglycosylase and a functionalizedxyloglucan oligomer comprising a chemical group; (h) a compositioncomprising a xyloglucan endotransglycosylase and a xyloglucan oligomer,and (i) a composition of (a), (b), (c), (d), (e), (f), (g), or (h)without a xyloglucan endotransglycosylase.

The inventions described and claimed herein are not to be limited inscope by the specific aspects herein disclosed, since these aspects areintended as illustrations of several aspects of the invention. Anyequivalent aspects are intended to be within the scope of theinventions. Indeed, various modifications of the inventions in additionto those shown and described herein will become apparent to thoseskilled in the art from the foregoing description. Such modificationsare also intended to fall within the scope of the appended claims. Inthe case of conflict, the present disclosure including definitions willcontrol.

1-19. (canceled)
 20. A modified agricultural crop comprising amodification selected from the group consisting of (a) a polymericxyloglucan and a functionalized xyloglucan oligomer comprising achemical group; (b) a polymeric xyloglucan functionalized with achemical group and a functionalized xyloglucan oligomer comprising achemical group; (c) a polymeric xyloglucan functionalized with achemical group and a xyloglucan oligomer; (d) a polymeric xyloglucan,and a xyloglucan oligomer; (e) a polymeric xyloglucan functionalizedwith a chemical group; (f) a polymeric xyloglucan; (g) a functionalizedxyloglucan oligomer comprising a chemical group; and (h) a xyloglucanoligomer, wherein the modified agricultural crop possesses an improvedproperty, and wherein the modified agricultural crop is a fruit, avegetable, a grain, a flower, or a spice.
 21. The modified agriculturalcrop of claim 20, wherein the improved property is one or moreimprovements selected from the group consisting of reducing orpreventing oxidative browning, dehydration, desiccation, bacterial,fungal, microbial, animal, or insect pest infestation, senescence, earlyripening, and softening; prevention of bruising, resistance to crushing,prevention or enhancement of clustering, aggregation, or association;resistance to adverse environmental factors; appearance, and taste, andresistance to sun or UV damage.
 22. The modified agricultural crop ofclaim 20, wherein the average molecular weight of the polymericxyloglucan or the polymeric xyloglucan functionalized with a chemicalgroup ranges from 2 kDa to about 500 kDa.
 23. The modified agriculturalcrop of claim 20, wherein the average molecular weight of the xyloglucanoligomer or the functionalized xyloglucan oligomer comprising a chemicalgroup ranges from 0.5 kDa to about 500 kDa.
 24. The modifiedagricultural crop of claim 20, wherein the polymeric xyloglucan or thepolymeric xyloglucan functionalized with a chemical group is present atabout 1 ng per g of the agricultural crop to about 1 g per g of theagricultural crop.
 25. The modified agricultural crop of claim 20,wherein the xyloglucan oligomer or the functionalized xyloglucanoligomer is present with the polymeric xyloglucan at about 50:1 molarratio to about 0.5:1 xyloglucan oligomer or functionalized xyloglucanoligomer to polymeric xyloglucan.
 26. The modified agricultural crop ofclaim 20, wherein the concentration of polymeric xyloglucan, thepolymeric xyloglucan functionalized with a chemical group, thexyloglucan oligomer, or the functionalized xyloglucan oligomercomprising a chemical group is about 0.01 g to about 500 mg per g of theagricultural crop.
 27. The modified agricultural crop of claim 20,wherein the xyloglucan oligomer or the functionalized xyloglucanoligomer is present without polymeric xyloglucan or polymeric xyloglucanfunctionalized with a chemical group at about 1 ng per g to about 1 gper g of the agricultural crop.
 28. The modified agricultural crop ofclaim 20, wherein the chemical group is a compound of interest or areactive group.
 29. The modified agricultural crop of claim 28, whereinthe reactive group is selected from the group consisting of an aldehydegroup, an amino group, an aromatic group, a carboxyl group, a halogengroup, a hydroxyl group, a ketone group, a nitrile group, a nitro group,a sulfhydryl group, and a sulfonate group.